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Neuroprotective effect of alcoholic extract of Terminalia arjuna
bark in experimental stroke induced rats
Thaakur Santhrani and Mangadevi Ch
Department of Pharmacology
Institute of Pharmaceutical Technology
Sri Padmavati Mahila Visvavidyalayam
Tirupati – 517502
INDIA
drsanthrani@gmail.com http://www.spmvv.ac.in
Abstract: - Terminalia arjuna Wight & Arn. (Combretaceae) is a tree having an extensive
medicinal potential and popular in various Indigenous Systems of Medicine like Ayurveda,
Siddha and Unani. Its stem bark possesses glycosides, large quantities of flavonoids, tannins and
minerals. Flavonoids are reported to exert antioxidant, anti-inflammatory and lipid lowering
effects. Evidence demonstrates that the impaired energy metabolism and the excessive generation
of reactive oxygen species (ROS) contribute to the brain injury associated with cerebral ischemia.
In the present study, the protective effect of T. arjuna bark alcoholic extract (TABE) was
investigated in transient middle cerebral artery occlusion (MCAO)-induced focal cerebral
ischemia–reperfusion injury in rats. Antioxidant activity of TABE was evaluated in vivo and in
vitro. Male albino rats were divided into five groups: Normal, Sham-operated group, Ischemic
control group, and TABE-pretreated groups (500 and 1000 mg/kg/p.o.). TABE was administered
once a day, for 14 days. The rats were subjected to a 2 h right MCAO via the intraluminal
filament technique and 22 h of reperfusion. Pretreatment with TABE significantly reduced the
histological changes and neurological deficits. TABE at a dose of 1000 mg/kg significantly
reversed the elevated total calcium levels, brain malondialdehyde (MDA) content and restored the
decreased activities of Na+-K
+ATPase, brain superoxide dismutase (SOD), catalase (CAT) and
reduced glutathione levels (GSH). TABE when tested against generation of oxygen free radicals
in vitro at the concentrations (64-1000 µg/ml), counteracted lipid peroxidation and the formation
of DPPH, nitric oxide (NO) and superoxide anions (O.2
-) with IC50 values 646.40 µg/ml, 488.18
µg/ml, 787.29 µg/ml and 1006.81 µg/ml respectively. Results of present study indicated that
TABE has a protective potential against cerebral ischemia injury and its protective effects may be
due to its antioxidant property.
Key-Words: Stroke, Ischemia, Cerebrovascular diseases, Oxidative stress, Terminalia arjuna,
Free radicals, Middle cerebral artery occlusion.
1 Introduction Stroke is an acute and progressive
neurodegenerative disorder. It is a medical
emergency and can cause permanent
neurological damage, complications and
death. Stroke is the second most common
cause of death worldwide [1] and the
leading cause of disease acquired adult
disability [2]. In western countries, stroke
causes 10-12% of all deaths [3], ranking
after heart disease and before cancer [4].
Until now there is no effective
neuroprotective therapy for the treatment of
stroke. Thus, there is an urgent need to
develop therapeutic strategies for the
management of stroke.
Ischaemic stroke reduces blood
supply, leads to energy shortage-induced
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imbalance of ionic gradients across
membrane, accumulates calcium, sodium,
and reduces pH, which in turn alters
membrane transport, mitochondrial function,
activates calcium-dependent enzymatic
reactions, including DNA-breaking enzymes
and generates excessive free radicals and
triggers lipid peroxidation and ultimately
cell apoptosis. Primary therapeutic strategy
in treating cerebral ischemia is to restore
blood flow in the shortest time possible in
order to preserve neural tissues. Although
reperfusion of ischemic brain tissue is
critical for restoring normal function, it can
paradoxically result in secondary damage,
called reperfusion injury. Secondary
approach is to ameliorate pathophysiological
consequences of stroke caused by free
radicals generated during ischemia and
reperfusion. Thus, antioxidants or free
radical scavenging agents are demonstrated
to have protective effect in experimental
stroke [5-10]. It is important to note that an
anti-ischemic agent aiming at both vascular
and parenchymal effects would be more
beneficial.
Terminalia arjuna (TA) is widely
used by Ayurvedic physicians for its
curative properties in organic/functional
heart problems including angina,
hypertension and deposits in arteries. Its
stem bark possesses glycosides, large
quantities of flavonoids, tannins and
minerals. Flavonoids have antioxidant, anti-
inflammatory and lipid lowering effects
while glycosides are cardiotonic. Flavonoids
and Oligomeric proanthocyanidins provide
antioxidant activity and vascular
strengthening [11]. It also has mild diuretic,
antithrombotic, prostaglandin E2 enhancing
and hypolipidaemic activity [12].
2 Materials and Methods
2.1 Plant material The wet bark of T. arjuna was collected
from Tirumala hills during the month of
November, 2009 and was shade dried. Stem
bark was coarsely powdered and refluxed
with 90% v/v methanol for six hours. The
extract was filtered and the filtrate was
evaporated to dryness by rotary flash
evaporator at low temperature under reduced
pressure. A dark brownish-red shiny powder
-like residue was obtained, which was stored
in a desiccator..
2.2 Animals Male albino rats weighing 200-250 g were
chosen to avoid fluctuations due to estrous
cycle. The rats were housed in
polypropylene cages under 12 hrs light /dark
cycle, fed with standard laboratory chow
(Hindustan Lever Limited, Mumbai) and
water ad libitum. Animals were acclimatized
to the laboratory conditions prior to
experimentation and all the experiments
were carried out according to the study
protocol approved by the Institutional
Animal Ethical Committee.
2.3 Induction of ischemia Transient focal cerebral ischemia was
produced by intra luminal middle cerebral
artery occlusion procedure as reported by
Longa et al.,[13] with slight modifications.
Briefly the rats were anaesthetized with
Thiopentone Sodium (40mg/kg, i.p.). A
midline incision was made on ventral side of
the neck and the right carotid bifurcation
was identified. The external carotid artery
was carefully isolated from accompanying
nerves and ligated. A small niche was given
to the right common carotid artery (CCA)
proximal to the carotid bifurcation and a 4-0
monofilament nylon thread (Ethicon,
Johnson & Johnson) with its tip rounded by
heating quickly by bringing it near a flame
was introduced through it into the lumen of
internal carotid artery and advanced until a
slight resistance was felt which ensured the
occlusion of the origin of middle cerebral
artery. Two hours after the induction of
ischemia, the filament was slowly
withdrawn and the animals were returned to
their cages for a reperfusion period of 22
hrs, the body temperature was maintained at
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370C with a thermostatically controlled
infrared lamp. In sham rats the right CCA
was surgically prepared for the insertion of
the filament, but the filament was not
inserted.
2.4 Study protocol Rats (200-250g) were randomly divided into
five groups of 9 rats in each group, fed with
drug or vehicle for 14 days prior to
experiment. The first group served as
normal and received 1% tween 80 in water
orally. The second group served as sham
control, received 1% tween 80 in water
orally. The third group served as ischemic
control, received 1% tween 80 in water
orally. The fourth and fifth groups were
treated with TABE 500 and 1000
mg/kg/p.o., respectively for 14 days prior to
MCAO. After 24 hrs, neurological
examinations were performed in all groups.
Then six rats in each group were decapitated
to obtain brain tissue samples for
biochemical analysis and remaining three
brains in each group were used for
histopathological studies.
2.5 Neurological deficit Neurological deficit in the vehicle- and
drug-pretreated groups were determined
after 24 hrs of induction. Neurological
findings were scored on a 5-point scale [13]
as follows: no neurological deficit = 0,
failure to extend left paw fully = 1, circling
to left = 2, falling to left = 3, did not walk
spontaneously and had decreased levels of
consciousness = 4. The above procedures
were performed in a blinded manner.
2.6 Biochemical estimations 24 h after induction of ischemia the animals
were killed by cervical dislocation and their
brains were dissected out quickly. The
infarcted area was homogenized in 50 mM
phosphate buffer (pH 7.0) containing 0.1
mM EDTA to give 5% w/v homogenate.
The homogenate was centrifuged at 10,000
rpm for 10 min at 0oC in cold centrifuge and
the resulting supernatant was used for
biochemical estimations.
2.6.1 Superoxide dismutase
SOD activity was measured according to the
method of Misra and Fridovich [14], at room
temperature. 100 µl of supernatant was
added to 880 µl of 0.05 M carbonate buffer
(pH 10.2, containing 0.1 mM EDTA), and
20 µl of 30 mM epinephrine (in 0.05%
acetic acid) was added to the mixture, and
the optical density values were measured at
480 nm for 4 min on a Systronics UV–
Visible Spectrophoto-meter; activity was
expressed as the amount of enzyme that
inhibits the oxidation of epinehrine by 50%
which is equal to 1 unit
.
2.6.2 Catalase
Catalase activity was measured by a slightly
modified version of Aebi, [15] at room
temperature. 100 µl of supernatant was
added to 10 ul of 100% ethanol and placed
in ice bath for 30 min. The tubes were
brought to room temperature followed by
the addition of 10 µl of Triton X-100. In a
cuvette containing 200 µl of phosphate
buffer and 50µl of above mixture, 250 µl of
0.066M H2O2 in phosphate buffer was
added. The decrease in optical density was
measured at 240 nm for 60 sec in Systronics
UV Spectrophotometer. The molar
extinction coefficient of 43.6 Mcm-1
was
used to determine catalase activity which is
equal to the moles of H2O2 degraded/mg
protein/min.
2.6.3 Reduced Glutathione
Reduced glutathione levels were measured
according to the method of Ellman [16] at
room temperature. 0.75 ml of supernatant
was mixed with 0.75 ml of 4%
sulphosalicylic acid and then centrifuged at
1200 rpm for 5 min at 4_C. From this 0.5 ml
of supernatant was taken and added to 4.5
ml of 0.01 M DTNB, and absorbance was
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measured at 412 nm by using a Systronics
UV–Visible Spectrophotometer.
2.6.4 Estimation of lipid peroxidation
MDA levels were measured according to the
method of Ohkawa et al., [17] at room
temperature. 200 µl of supernatant was
added to 50 µl of 8.1% sodium dodecyl
sulphate, vortexed, and incubated for 10 min
at room temperature. 375 µl of thiobarbituric
acid (0.6%) was added and placed in a
boiling water bath for 60 min and then the
samples were allowed to cool at room
temperature. A mixture of 1.25 ml of
butanol: pyridine (1.5:1) was added,
vortexed, and centrifuged at 1000 rpm for 5
min. The optical density values of colored
layer was measured at 532 nm on a Hitachi
U-2000 Spectrophotometer against reference
blank and the values were expressed in nM
of MDA formed for mg protein/min.
2.6.5 Estimation of total Calcium
To 4.5ml of deproteination buffer in a glass
centrifuge tube, 0.5ml of the sample was
added and was placed in water bath for
3minutes. Tubes were centrifuged while
they were still hot, 0.5ml of supernatant was
transferred into clean test tubes, 5ml of
working colouring reagent was added to
each tube, mixed well and then read at 570
nm. Calcium chloride solutions of 2-10 µg
calcium/ml were used for the preparation of
standard plot [18].
2.6.6 Estimation of Sodium potassium
ATPase (Na+-K
+ATPase) activity:
0.25 ml reaction mixture was taken in two
sets of test tubes. 0.1 ml of 10 mM Ouabain
was added to one set of tubes while to the
other, equal amounts of distilled water was
added. 50 µl of supernatant was added to
both the sets. Total volume of incubation
mixture was made upto 0.5 ml with the
addition of 50 µl of water to both sets of
test tubes (after preincubation period of 5
min), reaction was initiated with the addition
of 50 µl of ATP solution. The reaction was
terminated after 10 min by adding 0.5 ml of
10% TCA and the tubes were immediately
transferred to ice. Enzyme blank was run in
the similar way but supernatant was added
after the addition of TCA. Incubated in ice
for 10 min, all the tubes were centrifuged for
5 min to remove the precipitate. Whole
supernatant was taken for phosphate
estimation by micromethod. The enzyme
activity was expressed as µM of Pi
(inorganic phosphate) liberated / hr / mg
protein. The difference in the activity in the
absence and presence of Ouabain was taken
as Na+-K
+ATPase activity [19]. Activity in
the presence of Ouabain is Mg++ATPase
activity.
2.7 In vitro antioxidant studies
2.7.1 Anti lipidperoxidation assay
The extent of lipid peroxidation in rat brain
homogenate was measured in vitro in terms
of formation of thiobarbituric acid reactive
substances (TBARS). Different
concentrations of the TABE (64-1000
µg/ml) in ethanol were individually added to
the brain homogenate (0.5 ml). This mixture
was incubated with 0.15 M KCl (100 µl).
Lipid peroxidation was initiated by adding
100 µl of 15 mM FeSO4 solution and the
reaction mixture was incubated at 370C for
30 min. An equal volume of TBA
(thiobarbituric acid): TCA (trichloro acetic
acid), 1:1, 1 ml was added to the above
solution followed by the addition of 1 ml
butyrated hydroxyl toluene. This final
mixture was heated on a water bath for 20
min at 800 C, then cooled and centrifuged.
Absorbance of supernatent was read at 532
nm against control blank [20] using a
Systronics UV–Visible Spectrophotometer.
The percentage inhibition of lipid
peroxidation was calculated by using the
formula,
( )( )
100% xControl
TestControlInhibition
−=
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2.7.2 DPPH (1,1- diphenyl, 2-picryl
hydrazyl) radical scavenging activity
DPPH scavenging activity was measured by
the Spectrophotometric method [21]. To
ethanolic solutions of DPPH (200 µM), 0.05
ml of TABE dissolved in ethanol (63-1000
µg/ml) were added. After 20 min the
decrease in absorbance of test mixtures (due
to quenching of DPPH free radicals) was
read at 517 nm and the percentage inhibition
was calculated [20].
2.7.3 Scavenging of nitric oxide radical
Nitric oxide (NO) was generated from
sodium nitroprusside and measured by
Griess’ reaction [22, 23]. Sodium
nitroprusside (5 mM) in standard phosphate
buffer solution (0.025 M, pH: 7.4) was
incubated at 250C for 5 h with different
concentrations (64-1000 µg/ml) of TABE in
ethanol. Blank experiments without the test
compound but with equivalent amounts of
buffer were conducted in an identical
manner. After 5 h, 0.5 ml of incubation
solution was taken and diluted with 0.5 ml
of Griess’ reagent (1% napthyl ethylene
diamine dihydrochloride) and the
absorbance of the chromophore formed was
read at 546 nm and the percentage inhibition
was calculated [21, 24].
2.7.4 Scavenging of superoxide radical
The scavenging activity toward the
superoxide radical (O.2
-) was measured in
terms of inhibition of O2 [25] by following
alkaline dimethyl sulphoxide (DMSO)
method [26]. Potasssium superoxide and dry
DMSO were allowed to stand in contact for
24 h and the solution was filtered just before
use. The filtrate (200 µl) was added to 2.8
ml of an aqueous solution containing
nitroblue tetrazolium (56 µl), ethylene
diamine tetra acetic acid (10 µl) and
potassium phosphate buffer (10 mM ) then
the test compounds (1ml) at various
concentrations (64-1000 µg/ml) of TABE in
ethanol were added and absorbance was
recorded at 560 nm against a blank to
calculate the percentage inhibition.
2.8 Histopathology Coronal brain sections from normal and
experimental groups of focal ischemia were
fixed in a mixture of formaldehyde (4%),
glacial acetic acid, and methanol (1:1:8,
v/v). Brain slices were cut into 4- to 5-mm
thickness and embedded in paraffin blocks;
brain sections of 4 to 6 µm thickness were
stained with hematoxylin and eosin.
2.9 Statistical analysis All the data was expressed as mean ±
S.E.M, statsical significance was determined
by one way analysis of variance (ANOVA)
followed by Dunnet´s test, using GraphPad
prism 5. In all tests, the criteria for statistical
significance was P<0.05.
3 Results
3.1 Effect of TABE on neurological
deficit The neurological score after 22 hrs of
reperfusion was given in Table 1.
Pretreatment with TABE (500 and 1000
mg/kg/p.o.) significantly reduced the
neurological deficit (P<0.05 and P<0.001
respectively) [Fig. 1].
Table 1. Neurological scores
Values are expressed as Mean ± SEM (n=9);
*(p<0.05), ***(P<0.001) Vs Ischemic
control group.
Groups Neurological
score
Normal
Sham control
Ischemic control
TABE (500 mg/kg)
TABE (1000
mg/kg)
0
0
2.786 ± 0.2857
2.071 ± 0.1304*
1.571 ± 0.1304***
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3.2 Effect of TABE on brain MDA,
SOD, CAT and GSH levels The effect of TABE on brain MDA, SOD,
CAT, and GSH levels in MCAO-induced
ischemia-reperfusion rats is shown in Table
2. The content of brain MDA in the
ischemic hemisphere was significantly
increased (P<0.001) in Ischemic control
group as compared to the Normal group.
Pretreatment with TABE (500 and 1000
mg/kg/p.o.) significantly reversed the
elevated MDA levels (P<0.001) as
compared to the Ischemic control group
[Fig. 2].
The catalase level in the ischemic
hemisphere of Ischemic control group was
significantly decreased (P<0.001) as
compared to the Normal group. Pretreatment
with TABE (500 and 1000 mg/kg/p.o.)
significantly increased the decreased
catalase levels (P<0.001) when compared
with Ischemic control group [Fig. 4].
The level of reduced glutathione in the
ischemic hemisphere was significantly
decreased (P<0.001) in Ischemic control
group as compared to the Normal group.
Pretreatment with TABE (500 and 1000
mg/kg/p.o.) significantly increased the
reduced glutathione levels (P<0.001) when
compared with that of the Ischemic control
group [Fig. 5].
Table 2. Effect of TABE on tissue antioxidant parameters
Values are expressed as Mean ± SEM (n=6);
* (P<0.05) , ** (P<0.01), *** (P<0.001) Vs Normal group;
+++ (P<0.001) Vs Ischemic control group.
GROUPS
Superoxide
dismutase
(Units/mg protein)
Catalase
(µM/min/mg
protein)
Reduced
glutathione
(µM/mg protein)
MDA
(nM/mg protein)
Normal 1.596±0.05
51.32±0.96
0.097±0.01
0.261±0.02
Sham
control 1.519±0.08
50.47±0.06
0.088±0.01
0.228±0.01943
Ischemic
control 0.291±0.01***
15.43±1.87***
0.029±0.001***
0.674±0.01***
TABE
(500
mg/kg)
0.905±0.03***+++
45.66±0.99**+++
0.067±0.001**+++
0.463±0.01***+++
TABE
(1000
mg/kg)
1.333±0.02**+++
47.68±0.71+++
0.078±0.01*+++
0.306±0.01+++
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3.3 Effect of TABE on Calcium levels The effect of TABE on brain calcium levels
in different groups was given in Table 3.
The content of brain total calcium in the
ischemic hemisphere was significantly
increased (P<0.001) in Ischemic control
group as compared to the Normal group.
Pretreatment with TABE (500 and 1000
mg/kg/p.o.) significantly reversed the
elevated calcium levels (P<0.001) as
compared to the Ischemic control group
[Fig. 6].
3.4 Effect of TABE on Na+-K
+ATPase
activity Na
+-K
+ATPase activity in the brain tissues
of Ischemic control group rats was
significantly decreased (P<0.001) as
compared to that of the Normal group
(Table 3). Pretreatment with T. arjuna bark
extract (500 and 1000 mg/kg/p.o.)
significantly reversed the decrease in Na+-
K+ATPase activity (P<0.05 and P<0.001
respectively) as compared to the Ischemic
control group [Fig. 7].
3.5 In vitro antioxidant activity Several concentrations, ranging from 64-
1000 µg/ml of TABE in ethanol were found
to have a dose dependent free radical
scavenging activity in different in vitro
models like anti lipidperoxidation assay
(Fig. 8), DPPH scavenging activity (Fig. 9),
NO scavenging activity (Fig. 10) and
superoxide radical scavenging activity (Fig.
11). The maximum percentage inhibition at
1000 µg/ml concentration and IC50 values
are as given in Table. 4. On comparative
basis TABE showed excellent activity
against lipid peroxidation and in quenching
DPPH and nitric oxide radicals, but showed
moderate activity in quenching super oxide
radicals as given in Table 4.
Table 3. Effect of TABE on Calcium levels and Na+-K+ATPase activity
GROUPS
Calcium
(µg/mg protein)
Na+-K
+ATPase
(µM of Pi/h/mg protein)
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Values are expressed as Mean ± SEM (n=6);
* (P<0.05), *** (P<0.001) Vs Normal group;
+ (P<0.05), +++ (P<0.001) Vs Ischemic control group.
Table 4. In vitro antioxidant studies of TABE
In vitro model
% Inhibition
(at 1000 µµµµg/ml)
IC50
(µµµµg/ml)
Antilipid peroxidation assay
DPPH scavenging activity
NO scavenging activity
Superoxide scavenging activity
75.38
98.19
62.83
50.30
646.40
488.18
787.29
1006.81
Values are expressed as Mean ± SEM (n=6);
* (P<0.05) , ** (P<0.01), *** (P<0.001) Vs Normal group;
+ (P<0.05) , ++ (P<0.01), +++ (P<0.001) Vs Ischemic control group.
3.6 Effect of TABE on
histopathological studies Figures A and B show brain sections of
Normal and Sham control animals and
depicts normal cytoarchitecture of brain
tissue and neuronal cells (N) with normal
nucleus. Figure C show MCAO, hypo
perfusion-induced marked congestion of
blood vessels (HCBV), excessive
degeneration of neuronal cells (DN),
excessive vaculation (EV) and necrosis of
neural cells. Pretreatment with TABE 500
mg/kg showed mild congestion (MC), mild
degeneration (MD) of neuronal cells, and
mild vaculations (MV) in Fig D, where as
pretreatment with TABE 1000 mg/kg
showed neuronal cells with normal nucleus
(Fig E). Figures D and E showed that TABE
pretreatment dose dependently reversed the
congestion of blood vessels, congestion and
necrotic degeneration of neuronal cells
compared with Ischemic control group.
Normal 1.628±0.07603
0.440±0.02082
Sham control 1.569±0.05321
0.40±0.01291
Ischemic control
3.042±0.1077***
0.06667±0.007601***
TABE (500 mg/kg)
2.138±0.1941*+++
0.1217±0.01138***+
TABE (1000 mg/kg)
1.661±0.0380+++
0.2650±0.01057***+++
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4 Discussion
In the present study, pretreatment
with TABE significantly reversed the
MCAO-induced focal cerebral ischemia in
rats. Neurological deficit symptoms and
histopathological changes confirm the
evidence of the brain damage. Focal cerebral
ischemia causes cerebral cell death, resulting
in neuronal deficit symptoms and cerebral
infarction [27]. Numerous reports indicate
the involvement of oxidative stress in focal
cerebral ischemia [7, 8, 28]. In the present
study GSH, CAT, and SOD levels were
decreased and MDA level was increased,
indicating the involvement of oxidative
stress in MCAO induced reperfusion injury.
SOD dismutases superoxide
radicals to form hydrogen peroxide, which
in turn is decomposed to water and oxygen
by glutathione peroxidase and catalase,
thereby preventing the formation of
hydroxyl radicals [29]. Therefore, these
enzymes act co-operatively at different sites
in the metabolic pathway of free radicals.
Failure of this antioxidant defense leads to
oxidative damage and initiation of lipid
peroxidation [30]. Cerebral ischemia up
regulates inducible nitric oxide synthase
(iNOS), producing excessive nitric oxide
and it reacts with superoxide to form
peroxynitrite, which directly hydroxylate
and nitrate the aromatic residues of amino
acids and nucleotides in cytosol and nucleus
[31, 32], resulting in cellular machinery
dysfunction and neuronal loss after. Focal
cerebral ischemia increases MDA and
decreases GSH, CAT and SOD levels [7],
[8]. In the present study depletion of GSH
levels was observed in ischemic rats.
Reduced glutathione (GSH) is one of the
primary endogenous antioxidant defense
systems in the brain, which scavenges
hydrogen peroxide and lipid peroxides [33].
Depletion of GSH in ischemia reperfusion
injury is attributed to several factors such as
cleavage of GSH to cysteine, decrease in the
synthesis of GSH and the formation of
mixed disulfides, causing their cellular
stores to be depleted [34].
Excessive generation of ROS
results in the lipid peroxidation of the cell
membrane and subsequent damage is
reflected by accumulation of MDA, a
byproduct of lipid peroxidation [35]. Lipid
peroxidation is one of the major
consequences of free radical mediated injury
to the brain. The peroxidation of the
membrane phospholipids (PUFA) reaction
continues either until the exhaustion of the
substrate or termination of chain
propagation by antioxidants. Lipid
peroxidation products are widely accepted
group of oxidative stress markers, especially
MDA levels detected as a stable derivative
chromophore of Thiobarbituric acid adducts,
is used as a potentially reliable and sensitive
marker of reperfusion injury [36, 37].
Several studies demonstrated higher MDA
levels in stroke patients [38-42] and infarct
size, clinical severity and patient’s outcome
are well correlated with MDA levels [41],
[42]. Online with previous studies in the
present study, ischemic rats showed
significant increase in MDA levels
indicating reperfusion injury by ROS. Our
findings, viz. significantly elevated LPO and
depleted protective antioxidant enzymes
(GSH, CAT, and SOD) in
ischemia/reperfusion groups, are thus in
agreement with other reports on the role of
oxidative stress in cell injury in general and
cerebrovascular disease in particular [7, 43-
46]. In the present study, TABE exhibited
marked protection against MCAO induced
ischemia/reperfusion as evidenced by
significant reversal of enzymatic alterations
produced by such insult.
Ischemic cascade is initiated by
energy failure which quickly leads to
dysfunction of energy-dependent ion
transport pumps ( like Na+-K
+ATPase) and
depolarization of neurons and glia [47, 48].
Na+-K
+ATPase, which is responsible for the
maintenance of neuronal excitability and the
control of cellular volume in the central
nervous system [49], is an important
parameter to investigate stroke-induced
brain damage. Inhibition of the activity of
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this crucial enzyme is associated with
various neuropathological conditions,
including cerebral ischemia,
neurodegenerative disorders and spinal cord
edema, which also provide evidence for the
vulnerability of Na+-K
+ATPase to free
radical attacks [50-52]. In the present study,
decreased Na+-K
+ATPase activity due to
cerebral ischemia, indicates membrane
damage and deterioration of membrane
fluidity, is in accordance with increased
levels of calcium and lipid peroxidation.
TABE pretreatment (500 and 1000 mg/kg)
increased Na+-K+ATPase activity when
compared to Ischemic control group (P<0.5
and P<0.001). This effect of TABE may be
due to its powerful antioxidanat properties
thereby protecting membrane Na+-
K+ATPase from being attacked by the free
radicals produced during ischemia.
In the very early phase of ischemia,
membrane potential is lost due to energy
depletion consequently presynaptic neurons
and glia depolarize [53] to release
excitotoxic amino acids (especially
glutamate) into extracellular compartment in
large amounts. The activation of glutamate
receptors leads to a further increase of
intracellular Ca2+
, Na+ and Cl
- levels.
Calcium enters into damaged neurons
through voltage-gated calcium channels, via
N-methyl-D-aspartate (NMDA) receptor
operated channels, and release from
intracellular stores. Cytoplasmic calcium
activates enzymes and second messenger
cascades that contribute to cell death.
Activated proteolytic enzymes break down
elements of the cytoskeleton, leading to
protein aggregation. Calcium-mediated
lipolysis damages membranes and along
with nitric oxide synthase activation
provides nitric oxide and fatty acid
substrates for free radical production.
Glutamate release is stimulated by calcium-
dependent exocytosis and the released
glutamate in turn causes Ca2+
channels to
open, leading to further Ca2+
overload. In the
present study results demonstrated the
elevation of calcium levels in Ischemic
control group compared to Normal group
rats which is in consonance with the earlier
reports [54]. A significant decrease in total
calcium level observed in TABE
pretreatment groups may be due to either
calcium channel blocking or calcium
chelation or via antiglutaminergic activity of
some phytochemical constituent in the
extract. Free radicals are well documented to
increase calcium levels [55], [56], [57].
Therefore, the observed decrease in calcium
levels with TABE may also be attributed to
its anti-oxidant effects.
Search for agents providing
protection against lipid peroxidation and
enhancing anti-oxidant enzyme defense
system is a rational approach for therapy of
cerebrovascular ailments. Natural products
(i.e. medicinal plants) with intrinsic
antioxidant property constitute an ideal
choice for maximum therapeutic effects with
minimal risk of iatrogenic adverse effects.
Recently, several natural products like rutin,
sodium selenite, curcumin, isoliquiritigenin
(flavanoid), Spirulina, Acorus calamus
rhizomes extract, Thymoquinone, Nigella
sativa seed oil, and Korean ginseng tea
showed their protective effect on MCAO-
induced reperfusion injury due to their
antioxidant property [7, 8, 10,28, 58-60].
TA bark has active components
such as Arjunolic acid, tomentosic acid, β-
sitosterol, ellagic acid, (+)- leucodelphinidin
[64], arjunic acid [62]; arjunetin [63],
arjungenin, arjunglucoside I and II [65],
tannins containing catechin, gallocatechin,
epicatechin, epigallocatechin [66],
arjunolone, baicalein [67, 68],
arjunglucoside III [66], terminoic acid[69],
arjunolitin[70], arjunglucoside IV, V [71],
arjunasides A-E [72], 2α, 3β-dihydroxy urs-
12, 18 dien-28-oic acid 28-O-β-D-
glucopyranosyl ester [73], casuarianin [74],
arjunophthanoloside [76], terminoside A
[74], arjunin [77], terminarjunoside I, II
[78].
The phytochemical screening
of TABE showed the presence of
triterpenoids, tannins and flavonoids which
are arjunolic acid, arjunic acid, arjungenin,
arjunetin, arjunoglycoside I, arjunoglucoside
III, arjunoside I, arjunolitin, terminoside A,
arjunetoside, lupeol, arjunoglucoside II
Recent Researches in Modern Medicine
ISBN: 978-960-474-278-3 290
(triterpenoids), casuarinin, ellagic acid and
gallic acid (tannins), leucocyanidin and
luteolin (flavonoids). Tannins and
flavonoids are well known to have
antioxidant properties, anti-inflammatory
activity [79], antiproliferative as well as
anti-platelet effects [80, 81] and may be
responsible for significant rise in the
endogenous antioxidants (SOD, GSH and
CAT) of treatment groups in the present
study. Arjunolic acid (triterpene) a potent
principle isolated from TA has been shown
to prevent the decrease in levels of SOD,
CAT, GSH and ceruloplasmin ascorbic acid
and provided significant protection against
lipid peroxidation in Isoproterenol induced
myocardial necrosis in wistar rats [82].
Arjunic acid was significantly active against
the human oral (KB), ovarian (PA1) and
liver (HepG-2 and WRL-68) cancer cell
lines [83]. Luteolin, a flavone isolated from
the butanol fraction of TA was found to be
effective in inhibiting a series of solid
tumors (Renal A-549, ovary SK-OV-3,
Brain SF-295, Leukemia P 388, SKMEL-2,
XF-498, HCT 15, Gastric HGC-27).
Casuarinin, a hydrolysable tannin isolated
from the bark of TA protects cultured Madin
Darby canine kidney (MDCK) cells against
H2O2 mediated oxidative stress when
compared with Trolox (hydro soluble
Vitamin E analog) by decreasing DNA
oxidative damage and preventing the
depletion of intracellular Glutathione (GSH)
in MDCK cells [84]. Terminoside A isolated
from the acetone fraction of the ethanolic
extract of stem bark of TA potently inhibited
Nitric Oxide (NO) production and decreased
inducible Nitric Oxide Synthase (iNOS)
levels in lipopolysaccharide-stimulated
macrophages [75]. Arjunaphthanoloside
isolated from the stem bark of TA showed
potent antioxidant activity and inhibited
Nitric Oxide (NO) production in
lipopolysaccharide (LPS)-stimulated rat
peritoneal macrophages [76]. Arjungenin
and its glucoside isolated from the stem bark
of TA exhibited moderate free radical
scavenging activity in vitro and was found
to have moderate activity with an IC50 value
of 290.6 lg/ml as compared to standard
vitamin C [85]. These might contribute to
neuroprotection against cerebral ikschemia
too.
The results of present study
suggests that the TABE pretreatment dose
dependently protected the integrity of brain
tissue against MCAO-induced ischemia-
reperfusion injury as evidenced by
histopathological studies, neurological
deficit symptoms, brain SOD, CAT, GSH
levels, MDA content, total calcium levels
and Na+-K
+ATPase activity. Pretreatment
with TABE 500, 1000 mg/kg/p.o
significantly reversed and restored the levels
of SOD, GSH, CAT, Na+-K
+ATPase activity
and reduced LPO and calcium levels in
MCAO rats and there by prevented the
stroke induced changes in the brain to near
normal in the groups pre-treated with
TABE. The neuroprotective effect of TA
may be due to presence of flavonoids and
tannins.
In conclusion, these findings
suggest a potential role of Terminalia arjuna
in stroke and gain importance in view of the
fact that stroke is at present the third leading
cause of death worldwide, and
cerebravascular disease constitute second
most frequent cause of projected deaths.
TABE contains many active constituents
with potent antioxidant effectss. TABE
offered significant neuroprotection in
MCAO-induced focal cerebral ischemia, and
this may be attributed to inhibition of lipid
peroxidation and increase in endogenous
antioxidant defense enzymes. However,
further research is required for clinical use
of TA against stroke.
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Recent Researches in Modern Medicine
ISBN: 978-960-474-278-3 296
Histopathology
Fig A. Normal (magnification 40X)
Fig B. Sham control (Magnification 40X)
Fig C. Ischemic control (magnification 40X)
Fig D. TABE 500mg/kg pretreatment in
focal ischemia (magnification 40X)
N
N
N
HCBV
Nm
EV
DN
DN
MD
MC
MV
Recent Researches in Modern Medicine
ISBN: 978-960-474-278-3 297
Fig E. TABE 1000 mg/kg pretreatment in
focal ischemia (magnification 40X)
Fig. 1. Effect of TABE on Neurological
deficit in experimental stroke induced rats.
0
1
2
3
4
*
***
ISCHEMIC CONTROL TABE 500 mg/kg
TABE 1000 mg/kg
GROUPS
Neurological score
Values are expressed as Mean ± SEM (n=9);
* (p<0.05), *** (P<0.001) Vs Ischemic
control group.
Fig. 2. Effect of TABE on MDA levels in
experimental stroke induced rats.
0.0
0.2
0.4
0.6
0.8
***
+++
NORMAL SHAM ISCHEMIC CONTROL
TABE 500 mg/kg TABE 1000 mg/kg
+++***
GROUPS
nM
of M
DA/m
g pro
tein
Values are expressed as Mean ± SEM (n=6);
***(P<0.001) Vs Normal group;
+++ (P<0.001) Vs Ischemic control group.
Fig. 3. Effect of TABE on SOD levels
inexperimental stroke induced rats.
0.0
0.5
1.0
1.5
2.0
NORMAL SHAM ISCHEMIC CONTROL
TABE 500 mg/kg TABE 1000 mg/kg
***
***
**
+++
+++
GROUPS
Units/mg pro
tein
Values are expressed as Mean ± SEM (n=6);
**(p<0.01), ***(P<0.001)Vs Normal group;
+++ (P<0.001) Vs Ischemic control group.
Fig. 4. Effect of TABE extract on Catalase
levels in experimental stroke induced rats.
N
MD
N
Recent Researches in Modern Medicine
ISBN: 978-960-474-278-3 298
0
20
40
60
NORMAL SHAM ISCHEMIC CONTROL
TABE 500 mg/kg TABE 1000 mg/kg
***
** ++++++
GROUPS
µµ µµM
of H
2O
2/m
in/m
g pro
tein
Values are expressed as Mean ± SEM (n=6);
** (p<0.01), *** (P<0.001) Vs Normal;
+++ (P<0.001) Vs Ischemic control group.
Fig. 5. Effect of TABE on Glutathione
levels in experimental stroke induced rats.
0.00
0.05
0.10
0.15
NORMAL SHAM ISCHEMIC CONTROL
TABE 500 mg/kg TABE 1000 mg/kg
***
**
*
+++
+++
GROUPS
µµ µµM/mg protein
Values are expressed as Mean ± SEM (n=6);
* (p<0.05), ** (P<0.01), *** (P<0.001) Vs
Normal group; +++ (P<0.001) Vs Ischemic
control group.
Fig. 6. Effect of TABE on Calcium levels in
experimental stroke induced rats.
0
1
2
3
4
NORMAL SHAM ISCHEMIC CONTROL
TABE 500 mg/kg TABE 1000 mg/kg
***
+++
+++*
GROUPS
µµ µµg/m
g pro
tein
Values are expressed as Mean ± SEM (n=6);
* (p<0.05), *** (P<0.001) Vs Normal
group; +++ (P<0.001) Vs Ischemic control
group.
Fig. 7. Effect of TABE on Na+K+ATPase
activity in experimental stroke induced rats.
0.0
0.1
0.2
0.3
0.4
0.5
NORMAL SHAM ISCHEMIC CONTROL
TABE 500 mg/kg TABE 1000 mg/kg
***
***
***+++
+
GROUPS
µµ µµM
of Pi liber
ated/hr/
mg protein
Values are expressed as Mean ± SEM (n=6);
*** (P<0.001) Vs Normal group;
+ (P<0.05), +++ (P<0.001) Vs Ischemic
control group.
Recent Researches in Modern Medicine
ISBN: 978-960-474-278-3 299
In vitro studies
Fig. 8. Effect of TABE on Lipid
peroxidation.
Fig. 9. DPPH radical scavenging activity of
TABE.
Fig. 10. Nitric oxide scavenging activity of
TABE.
Recent Researches in Modern Medicine
ISBN: 978-960-474-278-3 300
Fig. 11. Superoxide radical scavenging
activity of TABE.
Recent Researches in Modern Medicine
ISBN: 978-960-474-278-3 301
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