Life Sciences 79 (2006) 1921 1928

www.elsevier.com/locate/lifescie

Effect of dietary sesame oil as antioxidant on brain hippocampus

of rat in focal cerebral ischemia
Saif Ahmad a,, Seema Yousuf a , Tauheed Ishrat a , M. Badruzzaman Khan a , Kanchan Bhatia b ,
Inayat Salem Fazli c , Jafar Salamat Khan d , Naseem Hasan Ansari e , Fakhrul Islam a,
a
Neurotoxicology Laboratory, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University),
New Delhi 110 062, India
b
Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
c
Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA
d
Council of Scientific and Industrial Research (CSIR), Ministry of Science and Technology, Govt. of India, Anusandhan Bhawan, Rafi Marg,
New Delhi 110 001, India
e
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 775550647, USA
Received 11 April 2006; accepted 13 June 2006

Abstract

Oxidative stress may be regarded as an imbalance between free radical production and opposing antioxidant defenses. Free radical oxidative
stress is implicated in rat cerebral ischemia and naturaceutical antioxidants are dietary supplements that have been reported to have neuroprotective
activity. Many studies have reported dietary sesame oil (SO) as an effective antioxidant. In the present study the neuroprotective effect of dietary
SO was evaluated against middle cerebral artery occlusion (MCAO)-induced cerebral ischemia injury in rats. Rats were fed on diet (20% SO) for
15 days. The middle cerebral artery of adult male Wistar rat was occluded for 2 h and reperfused for 22 h. The antioxidant properties of brain were
measured as levels of reduced glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxide (GPx), glutathione reductase (GR),
catalase (CAT), superoxide dismutase (SOD) and thiobarbituric acid reactive substance (TBARS). A decrease in the activity of all the enzymatic
and non-enzymatic antioxidants was observed along with an increase in lipid peroxidation (LPO) in MCAO group. The neurobehavioral activity
of rats was also observed by using videopath analyzer. Dietary SO improved the antioxidant status in MCAO + SO group when compared with
MCAO group. The results of neurobehavioral activity also support our biochemical data. The results obtained suggest protective effect of SO
against cerebral ischemia in rat brain through their antioxidant properties.
2006 Published by Elsevier Inc.

Keywords: Dietary sesame oil (SO); Cerebral ischemia; Hippocampus; Oxidative stress; Antioxidant; Neurobehavioral activity

Introduction causative mechanism suggested explaining this phenomenon is

In middle cerebral artery occlusion (MCAO)-induced cerebral
ischemia, various biochemical events occur that cause intracel-
lular calcium accumulation, depolarization, excessive release of
excitatory amino acids, especially glutamate and inhibition of
protein synthesis (Mies et al., 1993; Hossman, 1994). Cerebral
ischemia is one of the leading causes for several neurological
deficit and death (Chagnac-Amitai and Connors, 1989) and the
Corresponding authors. Tel.: +91 11 26059688; fax: +91 11 26059663.
E-mail addresses: saif_hamdard@yahoo.com (S. Ahmad),
fislam2001@yahoo.co.in (F. Islam).
0024-3205/$ - see front matter 2006 Published by Elsevier Inc.
doi:10.1016/j.lfs.2006.06.017
the involvement of reactive oxygen species (ROS) and oxidative
stress (Traystman et al., 1991; Wang and Lo, 2003; Blomgren et
al., 2003; Paradis et al., 2004). According to the literature, brain is
very vulnerable to oxidative stress due to its high polyunsaturated
fatty acids (PUFAs) content which are particularly susceptible to
ROS damage (Cui et al., 2004; Halliwell, 2001). During ischemia,
xanthine dehydrogenase undergoes irreversible proteolytic con-
version to xanthine oxidase, producing superoxide and hydrogen
peroxide in the presence of oxygen. Among the various free
radicals produced, superoxide and hydroxyl radicals are potent in
causing damage of cell membrane by inducing lipid peroxidation
(Bromont et al., 1989). Although NO (nitric oxide) functions as an
1922 S. Ahmad et al. / Life Sciences 79 (2006) 19211928
important neural messenger in the normal brain (Dawson et al.,
1992) however, on reperfusion of the ischemic brain, it reacts with
superoxide to form peroxinitrile which is a source of highly
reactive hydroxyl and other radicals (Bondy, 1995), hence playing
an important role in neuronal loss. Natural antioxidants have been
reported to protect brain damage against neurological diseases
(Thiyagrajan and Sharma, 2004; Anbarasi et al., 2005a,b; Ahmad
et al., 2005a,b).
Various dietary oils are currently receiving considerable
attention across the world for their potential health benefits in
relation to neurological disorders. SO (Sesame oil) is a
component of the traditional health food in India as well as in
various other oriental countries. The oil is effective against
various diseases including atherosclerosis, hypertension and
anti-aging effects (Fukuda et al., 1985, 1994; Namiki, 1995).
Along with sesaminol, which is the principal antioxidant
component in SO (Osawa et al., 1990), the lignans present
contribute towards its antioxidant and antimutagenic properties
(Kang et al., 1998a,b). SO contains a good amount of phenol,
sesamin, sesamol and sesamolin and relatively small amounts of
tocopherol which contributes to its superior oxidative stability
(White, 1992). In an earlier study, it was reported that SO
showed good antioxidant activity as compared to canola oil
(Baba et al., 1998). Sesame oil, in comparison to other dietary
oils such as ground nut and sunflower, offers better protection
against increased blood pressure, hyperlipidemia and lipid
peroxidation by increasing enzymatic and non-enzymatic
antioxidants (Sankar et al., 2005). Studies have demonstrated
SO and its active ingredient sesamol to be a strong antitumor
promoting agent when compared with resveratrol and sunflower
oil (Kapadia et al., 2002). The present study was undertaken to
observe the protective effect of dietary sesame oil against
oxidative stress in MCAO-induced focal cerebral ischemia in
rats.
Material and methods
Chemicals and drugs
Glutathione (oxidized and reduced), nicotinamide adenine
dinucleotide phosphate reduced (NADPH), ATP, qubain, 1-
chloro-2,4-dinitrobenzene (CDNB), 5-5-dithiobis-2-nitroben-
zoic acid (DTNB), thiobarbituric acid (TBA), 6-OHDA,
apomorphine hydrochloride, EDTA, NBT, 3,4-dihydroxyphe-
nyl acetic acid (DOPAC), 3,4-dihydroxybenzylamine (DHBA)
were purchased from Sigma Aldrich, USA, and other chemicals
were AR grade.
Dietary sesame seed oil
Sesame seed was provided by Grukul Kangari University,
Haridawar, Uttaranchal, India. SO was extracted from the
sesame seeds according to the method of Mohamed and Awatif
(1998). In brief, the sesame seeds were ground in a
disintegrator. The grounded seeds were extracted with n-hexane
for 48 h, and then filtered. This process was repeated three times
using fresh solvent each time to extract most of the oils from
ground seeds. Free fatty acids were determined by Gas Liquid
Chromatography (GLC) as per the protocol of Fazli et al. (2005)
and sesamin and sesamol content were analyzed according to
the method of Budowski et al. (1950) (Table 1).
Preparation of diet
Fat-free diet was provided by Animal House, Jamia
Hamdard, New Delhi. Sesame seed oil was mixed in the diet
in appropriate amount (200 ml SO [20%] was mixed in 1 kg
diet). Small pellets were prepared manually and dried at room
temperature.
Animals
Male Wistar rats weighing 300350 g were obtained from
the Central Animal House, Hamdard University, New Delhi.
Rats were housed in polypropylene cages in air-conditioned
room and were allowed free access to experimental and
pelletted diet (20% SO) and water ad libitum. The study was
approved by the Institutional Animal Ethics Committee (IAEC).
Experimental protocol
Animals were divided in four groups, each of six animals, for
15 days. The first group (sham) was fed pellet diet without
experimental diet; the second group was MCAO, i.e., ischemia
was induced for 2 h followed by reperfusion for 22 h; the third
group was fed experimental diet for 15 days followed by
MCAO for 2 h and reperfusion for 22 h (MCAO + SO group);
and the fourth group was fed experimental diet alone (20% SO
in diet) for 15 days. After the completion of the reperfusion
period, the animals were assessed for their neurobehavioral
activity and were sacrificed thereafter. The brains were taken
out and hippocampus was dissected for estimation of biochem-
ical parameters.
Induction of focal cerebral ischemia in rats
The right MCAO was produced using an intraluminal
filament model as described by Salim et al. (2003). In brief, the
Table 1
Analysis of fatty acid profile and Sesamin and Sesamolin content in Sesame seed
oil
Parameter Mean S.E.
Fatty acid (%)
Myristic acid C14:0 0.61 0.11
Palmitic acid C16:0 12.3 0.8
Stearic acid C18:0 3.9 0.2
Arachidic acid C20:0 0.23 0.07
Oleic acid C18:1 43.2 1.2
Linoleic acid C18:2 42.6 1.3
Linolenic acid C18:3 0.27 0.08
Seasamin (g/100 g) 0.8 0.06
Sesamolin (g/100 g) 0.5 0.04
Values are expressed as mean S.E.
 S. Ahmad et al. / Life Sciences 79 (2006) 19211928
rats were anesthetized with chloral hydrate (400 mg/kg, i.p.), a
40 nylon monofilament was inserted into the external carotid
artery and was advanced into the internal carotid artery. Two
hours after the induction of ischemia, the filament was slowly
withdrawn and the animals were put into their cages. In the
sham-operated rats, the external carotid artery was surgically
prepared for insertion of the filament but the filament was not
inserted.
Behavioral activity
The neurobehavioral activity in MCAO model of focal
cerebral ischemia was estimated by the slightly modified
method of Yamamoto et al. (1988). The behavioral activity was
monitored in video path analyzer (Coulbourn Instrument, USA)
by measuring the locomotion, average speed, rest and distance
travelled of the animal.
Tissue preparation
After producing MCAO and assessment of behavioral
activity, the animals were sacrificed immediately and their
brains were taken out to dissect the hippocampus. Post-
mitochondrial supernatant (PMS) obtained from 10% homo-
genate of tissue was used for the estimation of various
parameters related to the oxidative stress.
Biochemical estimation
Estimation of reduced glutathione (GSH). Reduced glu-
tathione was assayed by the method of Jollow et al. (1974).
One milliliter PMS (10%) was precipitated with 1.0 ml
sulfosalicylic acid (4%). The samples were kept at 4 C for
1 h and then subjected to centrifugation at 1200g for 15 min at
4 C. The assay mixture contained 0.1 ml filtered aliquot, 2.7 ml
phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (0.4% in
phosphate buffer 0.4 M, pH 7.4) in a total volume of 3.0 ml. The
yellow colour developed was read immediately at 412 nm. The
results were expressed as nmol GSH/g tissue.
Assay of lipid peroxidation (LPO). The procedure of Utley et
al. (1967) as modified by Islam et al. (2002) was used for the
estimation of the rate of lipid peroxidation. 1 ml of homogenate
(2.5% in chilled KCl) was pipetted in a glass radioimmunoassay
Table 2
Protective effect of SO on neurobehavioral activities in rat cerebral ischemia
Parameters Sham MCAO
Locomotor time (s) 189.33 9.15 105.5 10.11 (44.27%) a
Rest time (s) 150.67 10.68 222.83 10.97 (47.89%) a
Distance travelled (cm) 3574.5 261.69 1701.83 240.13 (52.38%) a
Average speed (cm/s) 604.5 40.4 342.83 31.8 (43.28%) a
Values are expressed as mean S.E.
p < 0.001 sham vs. MCAO; #p < 0.001 MCAO vs. MCAO + SO.
a
Values in parentheses indicate the percentage change vs. Sham.
b
Values in parentheses indicate the percentage change vs. MCAO.
1923
vial of 20 ml capacity and incubated at 37 1 C in a metabolic
shaker (120 rpm/min) for 60 min. Similarly, 1.0 ml of the same
homogenate was pipetted in a centrifuge tube and incubated at 0
C. After 1 h of incubation, 1.0 ml 5% chilled TCA was added
followed by 1.0 ml of 0.67% TBA in each vial and proper
mixing was done after each addition. The aliquot from each vial
was transferred to a centrifuge tube and centrifuged at 3500 rpm
for 15 min. Thereafter, supernatant was transferred to another
tube and placed in the boiling water bath. After 10 min, the test
tubes were cooled and the absorbance of the colour was read at
535 nm. The rate of LPO was expressed as nmol TBARS
formed/h/mg protein.
Estimation of glutathione-S-transferase activity (GST). Glu-
tathione-S-transferase (GST) activity was measured by the
method of Habig et al. (1974). The reaction mixture consisted
of 1.425 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml reduced
glutathione (1 mM), 0.025 ml CDNB (1 mM) and 0.3 ml
PMS (10%) in a total volume of 2.0 ml. The changes in
absorbance were recorded at 340 nm and enzyme activity was
calculated as nmol CDNB conjugate formed/min/mg protein
using a molar extinction coefficient of 9.6 103 M 1 cm 1.
Glutathione peroxide activity (GPx). GPx was estimated
according to the procedure described by Mohandas et al.
(1984). The reaction mixture consisted of 1.44 ml phosphate
buffer (0.05 M, pH 7.0), 0.1 ml EDTA (1 mM), 0.1 ml of
sodium azide (1 mM), 0.05 ml of glutathione reductase
(1 EU/ml), 0.1 ml of glutathione (1 mM), 0.1 ml of NADPH
(0.2 mM), 0.01 ml of hydrogen peroxide (0.25 mM) and
0.1 ml of PMS in the final volume of 2 ml. The
disappearance of NADPH at 340 nm was recorded at room
temperature. The enzyme activity was calculated as nmol
NADPH oxidized/min/mg/protein by using molar extinction
coefficient 6.22 103 M 1 cm 1.
Glutathione reductase activity (GR). Glutathione reductase
activity was assayed by the method of Carlberg and
Mannervik (1975), as modified by Mohandas et al. (1984).
The assay mixture consisted of 1.65 ml phosphate buffer
(0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM), 0.1 ml EDTA
(0.5 mM) and 0.05 ml oxidized glutathione (1 mM) and
0.1 ml of PMS in total volume of 2 ml. The enzyme activity
was quantitated at room temperature by measuring the
MCAO + SO SO alone
144.83 8.92#
(37.33%) b
194.0 12.21 (2.46%) a
186.83 8.12# (16.15%) b 167.67 10.14 (11.28%) a
2775.5 209.3# (63.08%) b 3130.0 201.6 (12.43%) a
490.07 29.69# (42.94%) b 575.33 35.60 (4.82%) a
1924 S. Ahmad et al. / Life Sciences 79 (2006) 19211928
Fig. 1. Protective effect of SO on the content of TBARS in the hippocampus.
Values are expressed as mean S.E. p < 0.001 sham vs. MCAO; ##p < 0.001
MCAO vs. MCAO + SO.
disappearance of NADPH at 340 nm and was calculated as
nmol NADPH oxidized/min/mg protein using molar extinc-
tion coefficient of 6.22 10 3 M 1 cm 1.
Catalase activity (CAT). Catalase activity was assayed by the
method of Caliborne (1985). Briefly, the assay mixture
consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0),
1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml PMS in total
volume of 3.0 ml. The change in absorbance was recorded at
240 nm. Catalase activity was calculated as nmol of H2O2
consumed/min/mg/protein.
Superoxide dismutase activity (SOD). SOD activity was
measured by the method of Beauchamp and Fridovich
(1971). The reaction mixture of total volume 1.0 ml
consisted of 0.5 M phosphate buffer pH 7.4, 0.1 ml PMS,
1.0 mM xanthine, 57 M NBT. It was incubated for 15 min
at room temperature and reaction was initiated by the
addition of 50 mU xanthine oxidase. The rate of reaction
was measured by recording change in the absorbance at
550 nm due to formation of formazan, a reduction product of
NBT.
Fig. 2. Protective effect of SO on the content of GSH (GSH/g tissue) in the
hippocampus. Values are expressed as mean S.E. p < 0.001 sham vs.
##
p < 0.001 MCAO vs. MCAO + SO.
Fig. 3. Protective effect of SO on the content of catalase in hippocampus. Values
are expressed as mean S.E. p < 0.001 sham vs. MCAO; ##p < 0.001 MCAO vs.
MCAO + SO.
Estimation of protein. Protein will be estimated by the method
of Lowry et al. (1951).
Statistical analysis. Results are expressed as mean S.E. of
six animals. Differences between the means of experimental and
control groups were analysed statistically by using Student's
t-test.
Results
The fatty acid and antioxidant profile of sesame oil is
discussed in Table 1. Similarly, the effect of MCAO and its
protection by dietary SO on locomotion, rest, distance travelled
and average speed is shown in Table 2. These neurobehavioral
activities were significantly (P < 0.001) decreased and rest time
was significantly increased in MCAO group as compared to
Sham group. The pretreatment with dietary SO in MCAO
group (MCAO + SO group) increased the locomotor time and
decreased the rest time as compared to MCAO group without
Fig. 4. Protective effect of SO on the content of SOD in hippocampus. Values are
expressed as mean S.E. p < 0.001 sham vs. MCAO; ##p < 0.001 MCAO vs.
MCAO + SO.
 S. Ahmad et al. / Life Sciences 79 (2006) 19211928
Table 3
Protective effect of Sesame oil (SO) on glutathione peroxide, glutathione reductase, glutathione-S-tranferase in rat cerebral ischemia
Enzymes Sham
GPx (nmol NADPH oxidized min 1 mg 1 protein) 17.17 1.02
GR (nmol NADPH oxidized min 1 mg 1protein) 37.4 1.58
GST (nmol CDNB conjugate formed min 1 mg 1 protein) 18.34 0.97
Values are expressed as mean S.E.
p < 0.001 sham vs. MCAO; #p < 0.001 MCAO vs. MCAO + SO.
a
Values in parentheses indicate the percentage change vs. Sham.
b
Values in parentheses indicate the percentage change vs. MCAO.
pretreatment with dietary SO. The Sham + SO group showed
no significant difference as compared to Sham without SO
group. The distance travelled (centimeters) was significantly
depleted in MCAO group as compared to Sham group. The
pretreatment with SO in group MCAO + SO showed significant
(P < 0.001) restoration of the distance travelled as compared to
MCAO group. However, the group Sham and Sham + SO
showed no significant effects. Significant (P < 0.001) results
were observed in average speed (cm/s) performed by MCAO
group due to the pretreatment of SO (MCAO + SO). The
average speed was significantly decreased in MCAO group as
compared to Sham group. No significant difference was
observed in Sham and Sham + SO group.
Figs. 1 and 2 show the effect of dietary SO on the TBARS
and GSH content in MCAO-induced cerebral ischemia. The
content of TBARS in hippocampus was significantly elevated
(P < 0.001) in MCAO group as compared to Sham group and its
content was significantly decreased in MCAO + SO group. The
level of GSH content was significantly depleted (P < 0.001) in
MCAO group as compared to Sham group and its content was
significantly attenuated in MCAO + SO group when compared
with MCAO group. No significant change was observed in SO-
alone group.
Figs. 3 and 4 show the effect of MCAO on the activity of
SOD and CAT and its protection by dietary SO in cerebral
ischemia. The activity of SOD and CAT were significantly
decreased in MCAO group (P < 0.001) as compared to Sham
group. These activities were observed to improve significantly
in MCAO + SO group as compared to MCAO group. These
activities were found to be of the same order in SO alone and
Sham groups.
Table 3 also shows the effect of MCAO on the activity of
GPx, GR and GST in hippocampus and protection offered by
dietary SO. The activities of GPx, GR and GST were found to
be significantly depleted in MCAO group (P < 0.001) as
compared to Sham. The activities of these enzymes were
significantly increased in MCAO + SO group as compared to
MCAO group. No significant change in the activities of these
enzymes was observed in the SO-alone group as compared to
Sham group.
Discussion
As shown in earlier studies, oxidative stress promotes lipid
peroxidation and alters the antioxidant defense system in brain
tissue under ischemic conditions (Thiyagrajan and Sharma,
1925
MCAO MCAO + SO SO alone
11.60 0.92 (32.44%) a 14.44 0.49### (24.48%) b 17.98 1.22 (4.71%) a
24.62 1.35 (34.17%)a 30.27 1.23## (22.94%)b 36.49 1.132 (2.43%) a
11.63 0.70 (36.78%)a 14.16 0.34# (21.75%)b 17.80 1.09 (2.94%) a
2004) Brain is highly susceptible to oxidative stress due to its
enrichment with non-heme iron that is catalytically involved in
the production of free radicals (Yousuf et al., 2005; Salim et al.,
2003). The plant-based antioxidants and edible oils have shown
to offer protection against human diseases as reported in many
studies (Ahmad et al., 2005a,b; White, 1992). Neuroprotective
effect of curcumin (Curcuma longa) has been reported
(Thiyagrajan and Sharma, 2004; Sharma et al., 2005). Cui et
al. (2004) reported the antioxidant activity of plant derived
active components (curcumin, turmerin, -carotene, lycopene
and lutin) in in vitro study. These antioxidant compounds were
found protective against DNA damage. In another study, the
memory enhancing and neuroprotective effect of Bacopa
monniera and its active ingredients bacoside A and bacoside
B have been reported (Tripathi et al., 1996; Anbarasi et al.,
2005a,b; Russo and Borrelli, 2005). Osawa et al. (1990)
reported that free radical-induced oxidative damage can be
effectively protected by use of various dietary antioxidants.
However, not many studies focused on neuroprotective effect of
dietary oils have been reported. In present study we used dietary
SO (20%) in MCAO-induced rat cerebral ischemia injury to see
its protective role and studied the antioxidant enzymatic and
non-enzymatic activities in brain hippocampus along with
behavioral activities. In our earlier study, the effect of dietary
SO (20%) was observed in the frontal cortex of rat brain in
MCAO-induced cerebral ischemia and significant enzymatic
and non-enzymatic activities were noted in MCAO + SO group
(Ahmad et al., 2005b).
The pretreatment with dietary SO before cerebral ischemia
for 15 days has shown significant improvement in locomotor
activity after 2 h of occlusion and 22 h of reperfusion (Table 2).
In the present study, it has been observed that dietary SO
significantly reduced lipid peroxidation level in brain hippo-
campus in MCAO + SO group as compared to MCAO group
(Fig. 1). The involvement of ROS and oxidative stress has
already been reported for damaging brain in MCAO-induced
cerebral ischemia and may contribute to selective neuronal
vulnerability of regions like hippocampus (Yousuf et al., 2005;
Gutsaeva et al., 2006). Impaired balance between prooxidant
and antioxidant mechanism results in lipid peroxidation (LPO)
which is a major cause for oxidative stress. The reduction of
LPO in MCAO + SO group is attributed to the presence of
lignans (sesamin, sesamol and sesamolin) in SO (Cooney et al.,
2001). Sesamin is reported to enhance hepatic detoxification,
reduce occurrence of chemically induced tumor, and protect
against oxidative stress (Akimoto et al., 1993; Hirose et al.,
1926 S. Ahmad et al. / Life Sciences 79 (2006) 19211928
1992). SO also contains vitamin E and is reported to increase
glutathione (Sharma et al., 2000), a reservoir of the aldehyde
binding compound cysteine. The presence of many antioxidant
enzymes such as SOD, CAT, GPx, GR and GST in the brain
prevents these tissues from the oxidative damage by free radical
formation (Cui et al., 2004). SOD reacts with the superoxide
radicals to form H2O2. CAT and GPx are involved in the
detoxification of H2O2, both at high and low concentrations. In
earlier studies, researchers have demonstrated that brain
contains low CAT levels, and hence, GPx has a major role in
quenching H2O2 and other peroxides which otherwise lead to
production of hydroxyl and peroxyl radicals in the presence of
iron (Bast and Barr, 1997; Gutteridge and Halliwell, 1994);
however, oxidative stress lowers these enzyme activities
(Ahmad et al., 2005a,b; Yousuf et al., 2005). GR is another
important enzyme for the maintenance of intracellular concen-
tration of reduced glutathione (GSH) (Gutteridge and Halliwell,
1994). Reduced glutathione serves as primary antioxidant
defense of brain against prooxidant stress and plays an
important role in behavioral activity (Cruz-Auguado et al.,
2001). The level of brain GSH is significantly reduced after
ischemic injury (Ravindranath and Reed, 1990). This depletion
was directly associated with elevation in brain lipid peroxida-
tion. The level of brain GSH has been attributed to offer
protection against ROS generation by ischemic stress besides its
consumption by the antioxidant enzymes, GPx and GST
(Baskaran et al., 1999). GST catalyses the detoxification of
oxidized metabolites of catecholamines (o-quinone) and may
serve as antioxidant, preventing degenerative processes (Baez et
al., 1997).
The SO mixed diet increased the antioxidant enzymes
activity in brain hippocampus in MCAO + SO group as
compared to MCAO group (Figs. 3, 4 and Table 3). This
increased activation of the antioxidant enzymes due to the
dietary SO could be due to the decreased utilization since the
LPO levels are low in the SO group. Our results are in
agreement with Prasanthi et al. (2005), as they have reported
increased antioxidant enzyme activity in oxidative damage in
rat tissue by using dietary SO. This is also supported by a wide
range of studies, which have shown that naturally occurring
antioxidants can prevent or slow down other diseases (Azuine
and Bhide, 1994; Gordon, 1996).
The exact mechanism by which dietary SO reduces oxidative
stress is not very clear from this study, but it is strongly believed
that the protective effect is due to the presence of lignans
(sesamin, sesamol and sesamolin) and vitamin E. The
antioxidant properties of lignans have already been shown in
several studies (Ogawa et al., 1995; Ide et al., 2001; Kapadia et
al., 2002; Abe et al., 2005). It has also been reported earlier that
vitamin E increases the glutathione (Sharma et al., 2000), a
reservoir for the aldehyde binding compound cysteine, and
significantly lowers blood pressure in rats (Newaz and Nawal,
1998). Vitamin E also lowers plasma lipid peroxides in diabetic
patients (Sharma et al., 2000). Furthermore, sesamol has also
been shown to be a classical inhibitor of LPO (Namiki, 1995;
Uchida et al., 1996). This study was also supported by the
significant improvement in behavioral activities in rats due to
the pretreatment of SO. These results clearly indicate a
correlation between the significant role of antioxidant enzymes
and non-enzymes on behavioral activities of rats as the
significant role of GSH has already been reported (Cruz-
Auguado et al., 2001).
Conclusion
In conclusion, the findings of our study clearly indicate
cerebral ischemia induced oxidative damage in brain in Male
Wistar rats in terms of neurobehavioral activities, increased
lipid peroxidation, depletion of glutathione level, and changes
in various enzymatic antioxidants. Our findings further
demonstrate that intake of dietary SO could effectively
ameliorate the cerebral ischemia-induced oxidative damage
which may be due to the presence of powerful antioxidant
compounds in sesame seed.
Acknowledgements
Dr. Saif Ahmad is thankful to the AYUSH (Ministry of
Health and Family Welfare, Government of India, New Delhi)
for providing research fellowship. Dr. S. N. Khan (DST, Govt.
of India) is gratefully acknowledged for her generous help and
valuable suggestions. The authors thank the lab attendants, Mr.
Dharamvir and Mr. Anil for the preparation of the diet, and
technical assistance. Conflict of Interest Statement: There is no
conflict of interest with any person or other organization.
References
Abe, C., Ikeda, S., Yamashita, K., 2005. Dietary sesame seeds elevate alpha-
tocopherol concentration in rat brain. Journal of Nutritional Science and
Vitaminology 51 (4), 223230.
Ahmad, M., Salim, S., Ahmad, A.S., Ansari, M.A., Yousuf, S., Hoda, M.N.,
Islam, F., 2005a. Neuroprotective effects of Withania somnifera on 6-
hydroxydopamine induced Parkinsonism in rats. Human and Experimental
Toxicology 24, 137147.
Ahmad, S., Ishrat, T., Badruzzaman, M.K., Yousuf, S., Ansari, M.A., Hoda,
M.N., Ahmad, A., Islam, F., 2005b. Neuroprotective effect of dietary
sesame oil, a natural antioxidant in cerebral ischemia. Symposium on
Emerging Trends in Neurosciences (XXIII Annual Meeting of Indian
Academy of Neurosciences at NIMHANS, Banglore, India): Abstract,
vol. 38, p. 54.
Akimoto, K., Kitagawa, Y., Akamatsu, T., Horose, N., Sugano, M., Shimizu, S.,
Yamada, H., 1993. Protective effects of sesamin against liver damage caused
by alcohol or carbon tetrachloride in rodents. Annals of Nutrition &
Metabolism 37, 218224.
Anbarasi, K., Vani, G., Balakrishna, K., Devi, C.S., 2005a. Effect of bacoside A
on membrane-bound ATPase in the brain of rats exposed to cigrette smoke.
Journal of Biochemical and Molecular Toxicology 19, 5965.
Anbarasi, K., Vani, G., Devi, C.S., 2005b. Protective effect of bacoside A on
cigrette smoking induced mitrochondrial dysfunction in rats. Journal of
Environmental Pathology, Toxicology and Oncology 24, 225234.
Azuine, M.A., Bhide, S.V., 1994. Adjuvant chemoprevention of experimental
cancer: catechin and dietary turmeric in forestomach and oral cancer models.
Journal of Ethanopharmacology 44, 211217.
Baba, N.H., Antoniades, K., Habbal, Z., 1998. Effects of dietary canola, olive,
and linolenic acid enriched olive oils on plasma lipids, lipid peroxidation and
lipoprotein lipase activity in rats. Nutrition Research 49, 4145.
Baez, S., Segura-Aguilar, J., Widersten, M., Johansson, A.S., Mannervik, B.,
1997. Glutathione transferases catalyse the detoxication of oxidized
 S. Ahmad et al. / Life Sciences 79 (2006) 19211928
metabolites (o-quinones) of catecholamines and may serve as an antioxidant
system preventing degenerative cellular processes. Biochemical Journal
324, 2528.
Baskaran, S., Lakshmi, S., Prasad, P.R., 1999. Effect of cigarette smoke on lipid
peroxidation and antioxidant enzymes in albino rat. Indian Journal of
Experimental Biology 37, 11961200.
Bast, A., Barr, P.R., 1997. The antioxidant/prooxidant balance in neurodegen-
eration and neuroprotection. In: Barr, P.R., Beal, M.F. (Eds.), Neuroprotec-
tion in CNS Disease. Marcel Deccer Inc., pp. 147159.
Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays
and an assay applicable to acrylamide gels. Annals of Biochemistry 44,
276287.
Blomgren, K., Zhu, C., Hallin, U., Hagberg, H., 2003. Mitrochondria and
ischemic reperfusion damage in the adult and in the developing brain.
Biochemical and Biophysical Research Communications 304, 551559.
Bondy, S.C., 1995. The relation of oxidative stress and hyperexitation to
neurological disease. Proceedings of the Society for Experimental Biology
and Medicine 208, 337345.
Bromont, C., Marie, C., Bralet, J., 1989. Increased lipid peroxidation in
vulnerable brain regions after transient forebrain ischemia in rats. Stroke 20,
918924.
Budowski, P., Connor, R.T., Field, E.T., 1950. Sesameoil: IV. Determination of
free and bound sesamol. Journal of the American Oil Chemists' Society 27,
307310.
Caliborne, A., 1985. Catalase activity. In: Wakd, G. (Ed.), CRC Hand Book
of Methods For Oxygen Radical Research. CRC Press, Boca Raton, FL,
pp. 283284.
Carlberg, I., Mannervik, B., 1975. Glutathione reductase levels in rat brain.
Journal of Biological Chemistry 250, 54755480.
Chagnac-Amitai, Y., Connors, B.W., 1989. Horizontal spread of synchronized
activity inneocortex by GABA-mediated inhibition. Journal of Neurophy-
siology 61, 747758.
Cooney, R.V., Custer, L.J., Okinaka, L., Franke, A.A., 2001. Effects of dietary
sesame seeds on plasma tocopherol levels. Nutrition and Cancer 39, 6671.
Cruz-Auguado, R., Almaguer-Melian, W., Diaz, M.C., Lorigados, L., Bergado,
J., 2001. Behavioural and biochemical effects of glutathione depletion in the
rat brain. Brain Research Bulletin 55, 327333.
Cui, Ke., Luo, X., Xu, K., Murthy, M.R.V., 2004. Role of oxidative stress in
neurodegeneration: recent developments in assay methods for oxidative
stress and Nutraceutical antioxidants. Progress in Neuro-Psychopharmacol-
ogy & Biological Psychiatry 28, 771799.
Dawson, T.M., Dawson, V.L., Snyder, S.H., 1992. A novel neuronal messenger
molecule in brain: the free radical, nitric oxide. Annals of Neurology 32,
297311.
Fazli, I.S., Abdin, M.Z., Jamal, A., Ahmad, S., 2005. Interactive effect of
sulphur and nitrogen on lipid accumulation, Acetyl-CoA concentration and
Acetyl-CoA carboxylase activity in developing seeds of oil seed crop
(Brassica campestris L. and Eruca sativa Mill.). Plant Science 168,
2936.
Fukuda, Y., Osawa, T., Namiki, M., Ozaki, T., 1985. Stidies on antioxidative
substancesin sesame seed. Agricultural and Biological Chemistry 49, 301306.
Fukuda, Y., Osawa, T., Namiki, M., 1994. In: Ho, C.T., Osawa, T., Huang, M.T.,
Rosen, R.T. (Eds.), Food Phytochemicals for Cancer Prevention II: Teas,
Spices and Herbs: A.C.S. Symposium Series, vol. 547, pp. 264274.
Gordon, M.H., 1996. Dietary antioxidants in disease prevention. Natural
Product Reports 13, 265273.
Gutsaeva, D.R., Suliman, H.B., Carraway, M.S., Demchenko, I.T., Piantadosi,
C.A., 2006. Oxygen-induced mitochondrial biogenesis in the rat hippo-
campus. Neuroscience 137, 493504.
Gutteridge, J.M.C., Halliwell, B., 1994. Antioxidant: elixirs of life or media
hype? Antioxidants in Nutrition, Health and Disease. Oxford University
Press, pp. 4062.
Habig, W.H., Pabst, M.J., Jokoby, W.B., 1974. Glutathione-S-transferase: the
first enzymatic step in mercapturic acid formation. Journal of Biological
Chemistry 249, 71307139.
Halliwell, B., 2001. Role of free radicals in the neurodegenerative diseases:
therapeutic implications for antioxidant treatment. Drugs and Aging 18,
685716.
1927
Hirose, N., Doi, F., Ueki, T., Akazawa, K., Chijiiwa, K., Sugano, M., Akimoto,
K., Shimizu, S., Yamada, H., 1992. Suppressive effect of sesamin against 7,
12-dimethylbenz- anthrecene induced rat mammary carcinogenesis.
Anticancer Research 12, 12591265.
Hossman, K., 1994. Viability thresholds and the penumbra of focal ischemia.
Annals of Neurology 36, 557565.
Ide, T., Ashakumary, L., Takahashi, Y., Kushiro, M., Fukuda, N., Sugano, M.,
2001. Sesamin, a sesame lignan, decreases fatty acid synthesis in rat liver
accompanying the down-regulation of sterol regulatory element binding
protein-1. BiochImica et Biophysica Acta 1534, 113.
Islam, F., Zia, S., Sayeed, I., Zafar, K.S., Ahmad, A.S., 2002. Selenium induced
alteration on lipids, lipid peroxidation, and thiol group in circadian rhythm
centers of rat. Biological Trace Element Research 90, 112.
Jollow, D.J., Mitchell, J.R., Zampaghone, N., Gillete, J.R., 1974. Bromoben-
zene induced oxide as the hepatotoxic intermediate. Pharmacology 11,
161169.
Kang, M.H., Katsuzaki, H., Osawa, T., 1998a. Inhibition of 2,2-azinobis(2,4-
dimethylvaleronitrile)-induced lipid peoxidation by sesaminols. Lipids 33,
10311036.
Kang, M.H., Naito, M., Tsujiohara, N., Osawa, T., 1998b. Sesamolin inhibits
lipid peroxidation in rat liver and kidney. Journal of Nutrition S128,
10181022.
Kapadia, G.J., Azuine, M.A., Tokuda, H., Takasaki, M., Mukainaka, T.,
Konoshima, T., Nishino, H., 2002. Chemopreventive effect of resveratrol,
sesamol, sesame oil and sunflower oil in the EpsteinBarr virus early
antigen activation assay and the mouse skin two-stage carcinogenesis.
Pharmacological Research 45 (6), 499504.
Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurement with Folin phenol reagent. Journal of Biological Chemistry
193, 265275.
Mies, G., Kohno, K., Hossman, K., 1993. MK-801, a glutamate antagonist,
lowers flow threshold for inhibition of protein synthesis after middle
cerebral artery occlusion of rat. Neuroscience Letters 155, 6568.
Mohamed, H.M.A., Awatif, I.I., 1998. The use of sesame oil unsaponifiable
matter as a natural antioxidant. Food Chemistry 62 (3), 269276.
Mohandas, J., Marshall, J.J., Duggin, G.G., Horvath, J.S., Tiller, D., 1984.
Differential distribution of glutathione and glutathione related enzymes in
rabbit kidneys: possible implication in analgesic neuropathy. Cancer
Research 44, 50865091.
Namiki, M., 1995. The chemistry and physiological function of sesame. Food
Reviews International 11 (2), 281329.
Newaz, M.A., Nawal, N.N.A., 1998. Effect of -tocopherol on lipid
peroxidation and total antioxidant atatus in spontaneously hypertensive
rats. American Journal of Hypertension 11, 14801485.
Ogawa, H., Sasagawa, S., Murakami, T., Yoshizumi, H., 1995. Sesame lignans
modulate cholesterol metabolism in the stroke-prone spontaneously
hypertensive rat. Clinical and Experimental Pharmacology & Physiology.
Supplement 22 (1), S310S312.
Osawa, T., Kumon, H., Namiki, M., Kawakishi, S., Fukuda, Y., 1990.
Antimutagenic heat stable anti-oxidants. Progress in Clinical and Biological
Research 342, 223238.
Paradis, E., Clavel, S., Julien, P., Murthy, M.R.V., de Bilbao, F., Arsenijevic, D.,
Giannakopoulos, P., Vallet, P., Richard, D., 2004. Lipoprotein lipase and
endothelial lipase expression in mouse brain: regional distribution and
selective induction following kainic acid-induced lesion and focal cerebral
ischemia. Neurobiology 15, 312325.
Prasanthi, K., Murlidhara, Rajini, P.S., 2005. Fenvalerate-induced oxidative
damage in rat tissues and its attenuation by dietary sesame oil. Food and
Chemical Toxicology 43, 299306.
Ravindranath, V., Reed, D.J., 1990. Glutathione depletion and formation of
glutathioneprotein mixed disulphide following exposure of brain mito-
chondria to oxidative stress. Biochemical and Biophysical Research
Communications 169, 10751079.
Russo, A., Borrelli, F., 2005. Bacopa monniera, a reputed nootropic plant: an
overview. Phytomedicine 12, 305317.
Salim, S., Ahmad, M., Zafar, K.S., Ahmad, A.S., Islam, F., 2003. Protective
effect of Nardostachys jatamansi in rat cerebral ischemia. Pharmacology,
Biochemistry and Behavior 74, 481486.
1928 S. Ahmad et al. / Life Sciences 79 (2006) 19211928
Sankar, D., Sambandam, G., Ramakrishna, Rao, Pugalendi, K.V., 2005.
Modulation of blood pressure, lipid profiles and redox status in hypertensive
patients takingdifferent edible oils. Clinica Chimica Acta 355, 97104.
Sharma, A., Kharb, S., Chugh, S.N., Kakkar, R., Singh, G.P., 2000. Evaluation
of oxidative stress before and after control of glycemia and vitamin E
supplementation in diabetic patients. Metabolism 49, 160162.
Sharma, R.A., Gescher, A.J., Steward, W.P., 2005. Curcumin: the story so far.
European Journal of Cancer 41, 19551968.
Thiyagrajan, M., Sharma, S., 2004. Neuroprotective effectof curcumin in middle
cerebral artery occlusion induced focal cerebral ischemia. Life Sciences 74,
969985.
Traystman, R.J., Kirsch, J.R., Koehler, R.C., 1991. Oxygen radical mechanisms
of brain injury following ischemia and reperfusion. Journal of Applied
Physiology 71, 11851195.
Tripathi, Y.B., Tripathi, E., Upadhyay, A., 1996. Antilipid peroxidative property
of Nardostachys jatamansi. Indian Journal of Experimental Biology 34,
11501151.
Uchida, M., Nakajin, S., Touoshima, S., Shinoda, M., 1996. Antioxidative effect
of sesamol and related compounds on lipid peroxidation. Biological &
Pharmaceutical Bulletin 19, 623626.
Utley, H.C., Bernhein, F., Hochslein, P., 1967. Effects of sulfhydryl reagent on
peroxidation in microsomes. Archives of Biochemistry and Biophysics 260,
521531.
Wang, X., Lo, E.H., 2003. Triggers and mediators of hemorrhagic transforma-
tion in cerebral ischemia. Molecular Neurobiology 28, 229244.
White, J.P., 1992. Fatty acids in oil seeds. Fatty Acids in Foods and Their Health
Applications. Dekker, M. Incorporated, New York, pp. 237262.
Yamamoto, M., Tamura, A., Kirino, T., Shimizu, M., Sano, K., 1988.
Behavioural changes after focal cerebral ischemia by left middle cerebral
artery occlusion in rats. Brain Research 452, 323328.
Yousuf, S., Salim, S., Ahmad, M., Ahmad, A.S., Ansari, M.A., Islam, F., 2005.
Protective effect of Khamira Abresham uood Mastagiwala against free
radical induced damage in focal cerebral ischemia. Journal of Ethnophar-
macology 99, 179184.