neuroprotective effect of alcoholic extract of terminalia arjuna … · 2011. 3. 1. · transient...

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

[email protected] 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

Recent Researches in Modern Medicine

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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


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


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.


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


decreased (P<0.001) in Ischemic control



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


* (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


increased (P<0.001) in Ischemic control



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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* (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


* (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


ISBN: 978-960-474-278-3 289

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


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.

5 References [1] Murray CJ and Lopez AD, Mortality

by cause for eight regions of the

world: Global Burden of Disease

Study, Lancet, Vol.349, 1997,

pp.1269–1276.

[2] Lees KR, Zivin JR, Ashwood T,

Davalos A, Davis SM, Diener HC,

Grotta J, Lyden P, Shuaib A,


ISBN: 978-960-474-278-3 291

Hardemark HP and Wasiewski

WW, Stroke-Acute Ischemic NXY

Treatment (SAINT I) Trial

Investigators. NXY-059 for acute

ischemic stroke, N Engl J Med,

Vol.354, 2006, pp.588–600.

[3] Bonita R, Epidemiology of stroke,

Lancet, Vol.339, 1992, pp.342–344.

[4] Donnan GA, Fisher M, Macleod M

and Davis SM, Stroke, Lancet,

Vol.371, pp.2001.

[5] Hwang IK, Yoo KY, Kim DS,

Jeong YK, Kim JD, Shin HK, Lim

SS, Yoo ID, Kang TC, Kim Dw,

Moon WK and Won MH,

Neuroprotective effects of grape

seed extract on neuronal injury by

inhibiting DNA damage in the

gerbil hippocampus after transient

forebrain ischemia, Life Sci, Vol.75,

2004, pp.1989–2001.

[6] Bhandari U and Ansari NM,

Protective effect of aqueous extract

of Embelia ribes Burm fruits in mid

cerebral ischemia in rats, Indian J

Pharmacol, Vol.40, 2008, pp.215–

220.

[7] Shah ZA, Gilani RA, Sharma P and

Vohora SB, Cerebroprotective effect

of Korean ginseng tea against global

and focal models of ischemia in rats,

J Ethnopharmacol, Vol.101, 2005,

pp.299–307.

[8] Thiyagarajan M and Sharma SS,

Neuroprotective effect of Curcumin

in middle cerebral artery occlusion

induced focal cerebral ischemia in

rats, Life Sci, Vol.74, 2005, pp.969–

985.

[9] Yanpallewar SU, Rai S, Kumar M

and Acharya SB, Evaluation of

antioxidant and neuroprotective

effect of Ocimum sanctum on

transient cerebral ischemia and long

term cerebral hypoperfusion,

Pharmacol Biochem Behav, Vol.79,

2004, pp.155–164.

[10] Hosseinzadeh H, Parvadeh S,

Nassiri M, Hamid R and Sadgnia

Ziaee T, Effect of Thymoquinone

and Nigella Sativa Seed oil on lipid

peroxidation level during global

cerebral ischemia reperfusion injury

in rat hippocampus, Phyto Med,

Vol.14, No.9, 2007, pp.621–627.

[11] Munasinghe TC, Seneviratne CK,

Thabrew MI and Abeysekera AM,

Antiradical and anilipoperoxidative

effects of some plant extracts used

by Sri Lanken traditional medical

practitioners for cardioprotection.

Phytother Res, Vol.15, 2001,

pp.519-523.

[12] Dwivedi S, Terminalia arjuna Wight

& Arn: A useful drug for

cardiovascular disorders, J

Ethnopharmacol, Vol.114,

2007, pp.114–129.

[13] Longa EZ, Weinstein PR, Carlson S

and Cummins R, Reversible middle

cerebral artery occlusion without

craniectomy in rats, Stroke, Vol.20,

1989, pp.84-91.

[14] Misra HP and Fridovich J, The role

of Superoxide anion in the

autooxidation of epinephrine and

simple assay for superoxide

dismutase, J Biol chem, Vol.247,

1979, pp.3170-3175.

[15] Aebi H, Catalase, Methods

Enzymology, Vol.105, 1984, pp.125-

126.

[16] Ellman GL, Tissue sulfhydryl

groups, Arch Biochem Biophys,

Vol.82, 1959, pp.70–77.

[17] Ohkawa H, Ohishi N and Yagi K,

Assay for lipid peroxides in animals

and tissue by thiobarbituric acid

reaction Ana Bhiochem, Vol.95,

1979, pp.351-358.

[18] Lorentz K, Improved determination

of serum calcium with

Orthocresolpthalein complexone,

Clinical Chem Acta, Vol.126, 1982,

pp.327-333.

[19] Reading HW and Isbir T, The role of

cation activated ATPase in

transmitter release from the rat iris,

Q J Exp Physiol, Vol.65, 1980,

pp.105-116.

[20] Kumar PV, Shashidhara S, Kumar

MM and Shridhara BY, Effect of


ISBN: 978-960-474-278-3 292

Luffa echinata on lipid peroxidation

and free radical scavenging activity,

J Pharm Pharmacol, Vol.52, 2000,

pp.891.

[21] Sreejayan N and Rao MNA, Nitric

oxide scavenging by curcuminoids,

J Pharm Pharmacol, Vol.49, 1997,

pp.105.

[22] Green LC, Wagner DA, Glogowski

J, Skipper PL, Wishnok JS and

Tannenbaum SR, Analysis of nitrate

and 15

N in biological fluids, Anal

Biochem, Vol.126, 1982, pp.131.

[23] Marcocci L, Maguire JJ, Droy-

Lefaix MT and Packer L, The Nitic

oxide scavenging property of Ginko

biloba extract 761, Biochem

Biophys Res Commun, Vol.201,

pp.748.

[24] Shirwaikar Annie and Somashekar

AP, Antiinflammatory activity and

free radical scavenging studies of

Aristolochia bracteolate Lam.,

Indian J Pharm Sci, Vol.65, 2003,

pp.68.

[25] Sanchez-Morena C, Methods used to

evaluate the free radical scavenging

activity in foods and biological

systems, Food Sci Tech Int, Vol.8,

2002, pp.122.

[26] Henry LEA, Halliwell B and Hall

DO, The Superoxide dismutase

activity of various Photosynthetic

organisms measured by a new and a

rapid assay technique, FEBS Lett,

Vol.66, 1976, pp.303.

[27] Park SJ, Nam KW, Lee HJ, Cho EY,

Koo U and Mar W, Neuroprotective

effects of an alkaloid free ethyl

acetate extract from the root of

Sophora flavescens Ait. against

focal cerebral ischemia in rats,

Phytomedicine, Vol.16, No.11,

2009, pp.1042-51.

[28] Zang C and Yang J, Protective

effects of isoliquiritigenin in

transient middle cerebral artery

occlusion induced focal cerebral

ischemia in rats, Pharmacol Res,

Vol.55, 2006, pp.303–309.

[29] Yao JK, Reddy R, Elhinny LG and

Vankammen DP, Effects of

haloperidol on antioxidant defense

system enzymes in schizophrenia, J

Psychiatr Res, Vol.32, 1998,

pp.385–391.

[30] Parikh V, Mohammad Khan M and

Mahadik SP, Differential effects of

antipsychotics on expression of

antioxidant enzymes and membrane

lipid peroxidation in rat brain, J

Psychiatr Res, Vol.37, 2003, pp.43–

51.

[31] Beckman JS, Beckman TW, Chen

J, Marshall PA and Freeman BA,

Apparent hydroxyl radical

production by peroxynitrite:

implications for endothelial injury

from nitric oxide and superoxide,

Proc Natl Acad Sci USA, Vol.87,

1992,pp.1620–1624.

[32] Moreno JJ and Pryor WA,

Inactivation of alpha 1-proteinase

inhibitor by peroxynitrite, Chem Res

Toxicol, Vol.5, 1992, pp.425–431.

[33] Coyle JT and Puttfarcken P,

Oxidative stress, glutamate and

neurodegenerative disorders,

Science, Vol.262, 1993, pp.689–

695.

[34] Shivakumar BR, Kolluri SV and

Ravindranath V, Glutathione and

protein thiol homeostasis in brain

during reperfusion after cerebral

ischemia, J Pharmacol Exp Ther,

Vol.274, 1995, pp.1167–1173.

[35] Halliwell B, Reactive oxygen

species in living system, source,

biochemistry and role in human

disease, Am J Med, Vol.99, 1991,

pp.145–225.

[36] Cano CP, Bermudez VP, Atencio

HE, Medina MT and Anilsa A,

Increased serum malondialdehyde

and decreased nitric oxide within 24

hours of thrombotic stroke onset,

Am J Ther, Vol.10, 2003, pp.473–

476.

[37] Soska V, Olsovsky J, Zechmeister

A, Lojek A, Bouda J and Garcia

Escamilla RM, Free oxygen radicals


ISBN: 978-960-474-278-3 293

and lipoperoxides in type 2 diabetic

patients, Revisions Mexicana

Pathologists Clinicians, Vol.44,

1997, pp.62–66.

[38] Re G, Azzimondi G, Lanzarini C,

Bassein L and Vaona I, Plasma

lipoperoxidative markers in

ischaemic stroke suggest brain

embolism, Eur J Emerg Med, Vol.4,

1997, pp.5–9.

[39] Bolokadze N, Lobjanidze I,

Momtselidze N, Solomonia R,

Shakarishvili R , Blood rheological

properties and lipid peroxidation in

cerebral and systemic circulation of

neurocritical patients, Clin

Hemorheol Microcir, Vol.30, 2004,

pp.99–105.

[40] Sharpe PC, Mulholland C and

Trinick T, Ascorbate and

malondialdehyde in stroke patients,

Ir J Med Sci, Vol.163, 1994,

pp.488–491.

[41] Polidori MC, Cherubini A, Stahl W,

Senin U and Sies H, Plasma

carotenoid and malondialdehyde

levels in ischemic stroke patients:

relationship to early outcome, Free

Radic Res, Vol.36, 2002, pp.265–

268.

[42] Gariballa SE, Hutchin TP and

Sinclair AJ, Antioxidant capacity

after acute ischaemic stroke, Q J M,

Vol.95, 2002, pp.685–690.

[43] Viglino P, Scara M, Rotilio G and

Rigo A, A kinectic study of the

reactions between H2O2 and Cu, Zn

superoxide dismutase, evidence for

an electrostatic control of the

reaction rate, Biochem Biophys

Acta, Vol.952, 1988, pp.77–82.

[44] Jenner P, Oxidative damage in

neurodegenerating diseases, Lancet,

Vol.344,1994, pp. 796–798.

[45] Farbiszewski R, Bielawski K,

Bielawska A and Sobaniec W,

Spermine protects in vivo the anti-

oxidant enzymes in transiently

hypoperfused rat brain, Acta

Neurobiol Expermentalis, Vol.55,

1995, pp.253–258.

[46] Delanty LN and Dichter MA,

Oxidative injury in the nervous

system, Arch Nerol Scand, Vol.98,

1989, pp.145–153.

[47] Martin RL, Lloyd HG and Cowan

AI, The early events of oxygen and

glucose deprivation: setting the

scene for neuronal death?, Trends

Neurosci, Vol.17, 1994, pp.251–

257.

[48] Katsura K, Kristian T and Siesjo

BK, Energy metabolism, ion

homeostasis and cell damage in the

brain, Biochem Soc Trans, Vol.22,

1994, pp.991–996.

[49] Streck EL, Feier G and Burigo M,

Effects of electroconvulsive seizures

on Na+-K+ATPase activity in the

rat hippocampus, Neurosci Lett,

Vol.404, 2006, pp.254-257.

[50] Grisar T, Glial and neuronal Na+-

K+ pump in epilepsy, Ann

Neuronal, Vol.16 Suppl, 1985,

pp.128-134.

[51] De Souza Wyse AT, Streck EL and

Worm, Preconditioning prevents the

inhibition of Na+,K

+-ATPase

activity after brain ischemia,

Neurochem Res, Vol.25, 2000,

pp.971-975.

[52] Yang YB and Piao YJ, Effects of

Resveratrol on secondary damages

after acute spinal cord injury in rats,

Acta Pharmavol Sin, Vol.24, 2003,

pp.703-710.

[53] Dirnagl U, Iadecola C and

Moskowitz MA. Pathophysiology of

ischaemic stroke: an integrated

view, Trends Neurosci, Vol.22

,1999, pp.391-397.

[54] Choi DW , Calcium-mediated

neurotoxicity: relationship to

specific channel types and role in

ischemic damage, Trends Neurosci,

Vol.11, 1988, pp.465-469.

[55] Okabe E, Odajima C, Taga R,

Kukreja RC, Hess ML and Ito H,

The effect of oxygen free radicals

on calcium permeability and

calcium loading at steady state in

cardiac sarcoplasmic reticulum,


ISBN: 978-960-474-278-3 294

Molecular Pharmacol, Vol.34,

1988, pp.388–394.

[56] Doan TN, Gentry DL, Tylor AA and

Elliott SJ, Hydrogen peroxide

activates agonist-sensitive Ca2+

influx pathways in canine venous

endothelial cells, The Biochemical

Journal, Vol.297, 1994, pp.209–

215.

[57] Kawakami M and Okabe E,

Superoxide anion radical-triggered

Ca2+ release from cardiac

sarcoplasmic reticulum through

ryanodine receptor Ca2+ channel,

Molecular Pharmacol, Vol.53,

1998, pp.497–503.

[58] Guptha R, Manjeet S and Ajay S,

Neuroprotective effect of

antioxidants on ischemia-

reperfusion induced cerebral injury,

Pharmacological research, Vol.48,

No.2, 2003, pp.209-215.

[59] Thaakur S and Sravanthi R ,

Neuroprotective effect of Spirulina

in cerebral ischemia–reperfusion

injury in rats, J Neuro Trans,

Vol.117, No.9, 2010, pp.1083-1091.

[60] Shukla PK, Khanna VK, Ali MM,

Mourya R, Khan MY and Srimal

RC, Neuroprotective effect of

Acorus Calamus against middle

cerebral artery occlusion induced

ischemia in rats, Hum Exp Toxicol,

Vol.25, 2006, pp.187–194.

[61] Rastogi RP and Mehrotra,

Compendium of Indian Medicinal

Plants, CSIR, New Delhi, Vol.1,

1993.

[62] Row LR, Murty PS, Subba-Rao

GSR, Sastry CSP and Rao K,

Chemical examination of

Terminalia arjuna: Part-XII:

Isolation and structure

determination of arjunetin from

Terminalia arjuna bark, Indian J

Chem, Vol.8, 1970, pp.772-775.

[63] Row LR, Murty PS, Subba Rao

GSR, Sastry CSP and Rao KVJ,

Chemical examination of

Terminalia arjuna: Part-XII:

Isolation and structure

determination of arjunic acid, a new

trihydroxytriterpene carboxylic acid

from Terminalia arjuna bark, Indian

J Chem, Vol.8, 1970, pp.716-721.

[64] Rastogi RP and Mehrotra BN,



1993.




1993.




1993

[67] Sharma PN, Shoeb A, Kapil RS

and Popli SP, Arjunolone: A new

flavone from stem bark of

Terminalia arjuna, Indian J Chem,

Vol.21B, 1982, pp.263-264.

[68] Sharma PV, Classical Uses of

Medicinal Plants, Chaukhambha

Visvabharati, Varanasi, India, 1996.

[69] Ahmad MU, Mullah KB, Norin T

and Ulla JK, Terminoic acid, a new

trihydroxytriterpene carboxylic acid

from bark of Terminalia arjuna,

Indian J Chem, Vol.22, 1983,

pp.738-740.

[70] Tripathi VK, PAndey VB, Udupa

KN and Rucker G, Arjunolitin, a

triterpene glycoside from

Terminalia arjuna, Phytochemistry,

Vol.31, 1992, pp.349-351.

[71] Wang W, Ali Z, Li XC, Shen Y

and Khan IA, Triterpenoids from

two Terminalia species, Planta

Med, Vol.76, No.15, 2010, pp.1751-

1754.

[72] Wang W, Ali Z, Li XC, Shen Y and

Khan IA, 18, 19-secooleanane type

triterpene glycosyl esters from the

bark of Terminalia arjuna, Planta

Med, Vol.76, No.9, 2010, pp.903-

908.

[73] Wang W, Ali Z, Li XC, Shen Y

and Khan IA, Ursane triterpenoids

from the bark of Terminalia arjuna,

Fitoterapia, Vol.81, No.6, 2010,

pp.480-484.


ISBN: 978-960-474-278-3 295

[74] Kuo PL, Hsu YL, Lin TC, Lin TT,

Chang JK and Lin CC, Induction of

cell cycle arrest and apoptosis in

human non-small cell lung cancer

A549 cells by casuarinin from the

bark of Terminalia arjuna Linn,

Anticancer Drugs, Vol.16, 2005,

pp.409-415.

[75] Ali A, Kaur G, Hamid H,

Abdullah T, Ali M, Niwa M and

Alam MS, Terminoside A, a new

triterpene glycoside from the bark of

Terminalia arjuna inhibits nitric

oxide production in murine

macrophages, J Asian Natl Prod

Res, Vol.5, 2003, pp.137-142.

[76] Ali A, Kaur G, Hayat K, Ali M and

Ather M, A novel naphthanol

glycoside from Terminalia arjuna

with antioxidant and nitric oxide

inhibitory activities, Pharmazie,

Vol.58, 2003, pp.932-934.

[77] Kandil FE and Nassar MI, A

tannin anti-cancer promotor from

Terminalia arjuna, Phytochemistry,

CSIR, New Delhi Vol.47, 1998,

pp.1567-1568.

[78] Alam MS, Kaur G, Ali A, Hamid

H, Ali M and Athar M, Two new

bioactive oleanane triterpene

glycosides from Terminalia arjun,

Natl Prod Res, Vol.22, 2008,

pp.1279-1288.

[79] Ishikawa Y, Sugiyama H, Stylianou

E and Kitamura M, Bioflavonoid

quercetin inhibits interleukin-1-

induced transcriptional expression

of monocyte chemoattractant

protein-1 in glomerular cells via

suppression of nuclear factor-B, J

Am Soc Nephrol, vol.10, 1999,

pp.2290–2296.

[80] Csokay B, Prajda N, Weber G and

Olah E, Molecular mechanisms in

the antiproliferative action of

quercetin, Life Sci, Vol.60, 1997,

pp.2157–2163.

[81] Landolfi R, Mower RL and Steiner

M, Modification of platelet function

and arachidonic acid metabolism by

bioflavonoids, Biochem Pharmacol,

Vol.33, 1984, pp.1525–1530.

[82] Sumitra M, Manikandan P, Kumar

DA, Natarajan A, Balakrishna K,

Manohar BM and Puvanakrishnan

R, Experimental myocardial

necrosis in rats: Role of arjunolic

acid on platelet aggregation,

coagulation and antioxidant status,

Mol Cell Biochem, Vol.277, 2001,

pp.135-142.

[83] Saxena M, Faridi U, Mishra R,

Gupta MM and Darokar MP,

Cytotoxic agents from Terminalia

arjuna, Planta Med, Vol.73, 2007,

pp.1486-1490.

[84] Chen CH, Liu TZ, Kuo TC, Lu FJ,

Chen YC, Chang-Chien YW and

Lin CC, Casuarinin protects

cultured MDCK cells from

hydrogen peroxide-induced

oxidative stress and DNA oxidative

damage, Planta Med, Vol.70, 2004,

pp.1022-1026.

[85] Pawar RS and Bhutani KK, Effect

of oleanane triterpenoids from

Terminalia arjuna-a

cardioprotective drug on the process

of respiratory oxyburst,

Phytomedicine, Vol.12, 2005,

pp.391-393.


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


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


* (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


***(P<0.001) Vs Normal group;


Fig. 3. Effect of TABE on SOD levels

inexperimental stroke induced rats.

0.0

0.5

1.0

1.5

2.0



***

***

**

+++

+++

GROUPS

Units/mg pro

tein


**(p<0.01), ***(P<0.001)Vs Normal group;


Fig. 4. Effect of TABE extract on Catalase

levels in experimental stroke induced rats.

N

MD

N


ISBN: 978-960-474-278-3 298

0

20

40

60



***

** ++++++

GROUPS

µµ µµM

of H

2O

2/m

in/m

g pro

tein


** (p<0.01), *** (P<0.001) Vs Normal;


Fig. 5. Effect of TABE on Glutathione

levels in experimental stroke induced rats.

0.00

0.05

0.10

0.15



***

**

*

+++

+++

GROUPS

µµ µµM/mg protein


* (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



***

+++

+++*

GROUPS

µµ µµg/m

g pro

tein


* (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



***

***

***+++

+

GROUPS

µµ µµM

of Pi liber

ated/hr/

mg protein


*** (P<0.001) Vs Normal group;

+ (P<0.05), +++ (P<0.001) Vs Ischemic

control group.


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.


ISBN: 978-960-474-278-3 300

Fig. 11. Superoxide radical scavenging

activity of TABE.


ISBN: 978-960-474-278-3 301

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