Auroma Ghoshet al.

/ Journal of Pharmacy Research 2015,9(2),105-118

Research Article
ISSN: 0974-6943

Available online through

www.jpronline.info

Melatonin affords protection against myocardial

ischemia-induced cerebral mitochondrial dysfunction: an in vivo study.
Auroma Ghosh1, Mousumi Dutta1,2, Arnab Kumar Ghosh1, Aindrila Chattopadhyay2, Debajit Bhowmick3 and Debasish Bandyopadyay1*
Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata 700 009, India
2
Department of Physiology, Vidyasagar College, 39 Sankar Ghosh Lane, Kolkata 700 006, India
3
Acharaya Prafulla Chandra Sikhsha Prangan, University of Calcutta, JD-2, Sector-III, Salt Lake City, Kolkata 700 098, India

Received on:28-12-2014; Revised on: 17-01-2015; Accepted on:23-02-2015

ABSTRACT
Background: Ischemic Heart Disease (IHD) is a health problem of global concern. The studies on myocardial ischemia induced changes in
brain particularly cerebrum portion are scanty. Lacunae exist in the knowledge whether such changes can be protected by antioxidant(s). The
present work was carried out to explore the ameliorating potential of melatonin against isoproterenol induced changes in tissue and mitochondria isolated from heart and brain of male Wistar rats. Methods: The adverse changes were induced by administering isoproterenol
bitartrate subcutaneously at a dose of 25mg/kg body weight. Protective effects of melatonin was examined by administering melatonin at the
dose of 40mg / kg body weight. After the treatment period, biomarkers of organ damage and oxidative stress biomarkers, activities of
mitochondrial antioxidant as well as Krebs cycle enzymes, and tissue and mitochondrial morphology was studied. Results: Isoproterenol
administration caused remarkable deleterious changes in oxidative stress biomarkers like lipid peroxidation level, reduced glutathione and
protein carbonyl content of tissue and mitochondria. Moreover, isoproterenol unfavorably altered the activity of antioxidant enzymes like
Mn-SOD, glutathione peroxidase (GPx) and glutathione reductase (GR) in mitochondria thus confirming the generation of oxidative stress in
mitochondria. Altered activity of some of the important Krebs cycle enzymes indicated the disturbance in energy metabolism. The degeneration of mitochondria was observed by scanning electron microscopy and fluorescence confocal microscopy. Melatonin pre-treatment
successfully restored all the parameters to normal level at a dose of 40mg/kg body weight. Conclusion: Isoproterenol causes deleterious
changes in cardiac and cerebrum portion of the brain tissue as well as the mitochondria and melatonin pre-treatment protects against such
changes. Thus, therapeutic use of melatonin may be considered in treating cardiovascular diseases along with subsequent brain damages as
there may be strong relationship between cardiovascular diseases and cognitive functioning of brain.
KEYWORDS: Brain, heart, isoproterenol, melatonin, mitochondria, oxidative damage
INTRODUCTION:
A large number of people are still suffering from myocardial ischemia
in spite of modern health care facilities and well known therapeutic
measures1. So, it is a real concern for researchers, medical and health
professionals to counteract the insults on heart arising from myocardial ischemia. In myocardial ischemia there is prolonged insufficient
oxygenated blood supply to heart leading to the generation of reac*Corresponding author.
Dr. Debasish Bandyopadhyay
Professor
Oxidative Stress and Free Radical Biology Laboratory
Department of Physiology, University of Calcutta
92, APC Road, Kolkata 700 009, India
Principal Investigator, Center with Potential for Excellence in a
Particular Area (CPEPA)
University of Calcutta, 92 APC Road, Kolkata 700 009, India

tive oxygen species (ROS) and reactive nitrogen species (RNS) which
ultimately give rise to oxidative stress and myocardial damage2, 3. Disruption in normal functioning of heart can trigger a potential threat to
other organs. Many recent studies indicated that there may be strong
relationship between cardiovascular diseases and cognitive impairment4 though further investigations are needed as cognitive changes
are multi-factorial. According to elementary physiological knowledge
it is apprehensible that insufficient blood supply to brain due to myocardial ischemia may cause oxidative damages of brain but specific
physiological and biochemical pathways involved in this association
are yet to be determined.
Oxidative stress is one of the prime reasons for damage of different
organs5. It occurs when there is a disproportion between the antioxi-

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dant defense mechanisms and free radical generation. Excessive free
radical accumulation leads to distortion of different vital molecules
like DNA, proteins, lipids etc. Mitochondria being the energy generator of the cell is highly susceptible to oxidative stress. During ATP
synthesis electrons are transported through different complexes of
electron transport chain (ETC) present in inner membrane of mitochondria. These electrons are finally transported to oxygen to produce water and if sufficient amount of oxygen is not available then
this transportation of electron will generate reactive intermediates
which will cause damage to biomacromolecules. During this electron
transport single electron leaks out and reacts with oxygen molecule
thus generating ROS 6. Excessive amount of ROS will be accumulated
in the system if they are not neutralized and/ or scavenged immediately by antioxidant defense mechanisms of cell. Reactive oxygen
species, particularly, hydrogen peroxide (H2O2) can easily react with
iron to produce extremely reactive hydroxyl radical (OH) which in
turn destroys the essential macromolecules like proteins, lipids and
nucleic acids2..
Isoproterenol (ISO) is a synthetic catecholamine and also known as
a beta adrenergic receptor agonist. It is widely used to produce myocardial ischemia and myocardial infarction in experimental animal
models. Many studies have indicated that in rat heart ISO can produce similar changes that are generally seen in human heart after
myocardial infarction. It has been reported that ISO initiates oxidative
stress which ultimately produces myocardial ischemia and myocardial injury2, 7.
It is well evidenced that melatonin (N-acetyl-5- methoxytryptamine)
is a potent antioxidant and free radical scavenger8. Many studies
have demonstrated the protective effects of melatonin against myocardial infarction2, 9. It gives protection by diminishing the ROS level
by donating electrons to free radicals10. The present study has attempted to explore not only the extent of protection that melatonin
affords against isoproterenol induced myocardial injury but also to
determine the effect of myocardial ischemia on cerebrum portion of
the brain at the level of mitochondria and has also investigated and
gauged the efficiency of melatonin to favorably protect the effects of
myocardial ischemia on brain.
METHODS AND MATERIALS:
Chemicals and reagents:
Melatonin and isoproterenol bitartrate were procured from Sigma
Aldrich,USA. The other chemicals and reagents used in this study
were of analytical grade and procured from Sisco Research Laborato

ries (SRL), Mumbai, India, Qualigens (India/Germany), Merck Limited, Delhi, India and SD fine chemicals ,India.
Animals:
The study protocol had been approved under the Centre with Potential for Excellence in Particular Area (CPEPA) Scheme by the Institutional Animal Ethics Committee (IAEC) of the Department of Physiology, University of Calcutta. The CPCSEA registered animal supplier
was selected for procurement of male wistar rats, weighing 150-190g.
The guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) for animal handling
were strictly followed during the experiment tenure.
Experimental Design:
All rats were supplied with water ad libitum and food during the
experiment and quarantine periods. The rats were divided into 4
groups:
1.
2.

3.

4.

Control: This group comprised of vehicle treated rats.

Positive Control: The rats in this group were injected i.p.
with melatonin at the dose of 40mg/kg body weight, one
injection 24 hour apart, i.e., for 2 days.
Treated Group: Isoproterenol bitartrate (ISO) was injected
sub-cutaneously to this group of rats at the dose of 25 mg/
kg body weight, one injection 24 hour apart, i.e, for 2 days.
Protected Group: Rats in this group were injected i.p. with
40 mg/kg body weight melatonin, 30 min prior to ISO injection (25 mg/ kg body weight, s.c.); two injections of each 24
hour apart, i.e., for 2 days.

The animals were kept in a room of animal house where temperature

(251 0C), humidity (50 10%) and 12 hr light/dark cycle were maintained. The tenure of the treatment was 2 days.
Animal sacrifice and collection of blood and tissue samples:
The animals were sacrificed through cervical dislocation following
mild ether anesthesia after the stipulated time period that is 24 hr after
the last ISO injection. The chest cavity was surgically opened. Then
blood was collected carefully through cardiac puncture for serum
preparation and after blood collection the heart was surgically extirpated. The whole brain is collected by carefully opening the cranial
cavity and cerebrum portion was separated. The rinsing of the tissues was done in cold saline. After rinsing, the tissues were soaked
properly with blotting paper and stored at -200C in sterile plastic vials
for biochemical analysis. A small portion of the tissue was fixed in
appropriate fixative for tissue morphological studies.
Measurement of level of activity of SGOT:
Serum glutamate oxaloacetate transaminase (SGOT) was measured

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by applying standard protocols. The obtained values were expressed
as IU/L.11
Preparation of heart and brain mitochondria:
The procedure of Dutta et al.12 was followed to separate mitochondria
from heart and brain tissue homogenates. Briefly, 10% cardiac and
brain tissue homogenates were prepared separately in a Potter
Elvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA) by
using ice cold sucrose buffer containing 0.25 (M) sucrose, 0.001(M)
EDTA, 0.05(M) Tris-HCl (pH 7.8) at 250C. The homogenates were
then centrifuged at 500 g for 10 mins under cold condition i.e at 4 0C.
The supernatants, thus obtained, were again centrifuged at 12000 g
for 15mins under cold condition. The obtained pellet containing mitochondria was re-suspended in sucrose buffer and stored at -200C for
biochemical assays.
Measurement of lipid peroxidation (LPO) level, reduced glutathione
(GSH) and protein carbonyl (PCO) content of cardiac and brain tissues and mitochondrial fraction:
Ten percent tissue homogenates were prepared in ice-cold 0.9% saline. Lipid peroxides in the whole homogenates and mitochondrial
samples were estimated as thiobarbituric acid reactive substances
(TBARS) by following the method of Buege and Aust 13 with some
modification as adopted by Bandyopadhyay et al.14. Thiobarbituric
acid-trichloro acetic acid (TBA-TCA) reagent was mixed to measured
amount of tissue homogenates and mitochondrial samples separately
with thorough shaking and then the mixtures were heated at 800C for
20 min. The samples were then cooled to room temperature and centrifuged. The absorbance of the pink chromogen present in the supernatant was recorded spectrophotometrically at 532 nm using a UV
VIS spectrophotometer (Shimadzu). Values were determined as
nmoles/mg of protein.
To estimate reduced glutathione (GSH) content, the method of Sedlac
and Lindsay 15 along with some modifications 14 was used (i.e., by
using Ellmans reagent). Briefly, 10% tissue homogenates were prepared by using 2mM ice cold ethylene diamine-tetraacetic acid
(EDTA). Tris-HCl buffer (pH 9.0) was then mixed with the
homogenates and previously prepared mitochondrial samples. Finally, DTNB was added to the above samples for color development
and the absorbance was noted spectrophotometrically at 412 nm.
Reduced glutathione content of the samples were expressed as nmoles/
mg protein.
The DNPH assay16 was used to determine the protein carbonyl (PCO)
content of both the tissue and the mitochondria. Tissue homogenates
were prepared by same procedure as mentioned in the measurement

of LPO. Briefly, 10 mM DNPH dissolved in 2(N) HCl was added to the

measured amount of mitochondrial suspension and or tissue homogenate. Then, the samples were kept in dark at room temperature for 1
hr with occasional mixing. After the incubation period, proteins of the
samples were precipitated by the addition of 30% TCA followed by
centrifugation at 4000g for 10 mins. The excess DNPH present in the
pellet was washed with ethanol: ethyl acetate (1:1,v/v) mixture. This
washing was done for three times and after final washing the pellet
was dissolved in 6(M) guanidine hydrochloride prepared in 20mM
potassium dihydrogen phosphate (pH2.3). The absorbance was recorded spectrophotometrically at 370 nm. The obtained values were
expressed as nmoles/ mg of protein.
Determination of the activities of antioxidant enzymes present in
mitochondria:
Pyrogallol autooxidation method 17 was used to determine the manganese superoxide dismutase (Mn-SOD) activity. Increase in absorbance was noted at 420nm for 3min in a UV/VIS spectrophotometer
(Smart Spec Plus; BioRad). Activity of the enzyme was expressed as
units/min/mg of protein.
Method of Paglia and Valentine 18 with some modifications adopted
by Chattopadhyay et al. 19 was applied to assess the glutathione
peroxidase (GPx) activity. Final volume of 1 ml assay mixture consisted of 0.05 M phosphate buffer with 2mM EDTA pH7.0, 0.025mM
sodium azide, 0.15mM glutathione, 0.25mM NADPH and mitochondrial sample as enzyme source. The H2O2 (0.36 mM) was added to
initiate the reaction. Decrease in absorbance was noted spectrophotometrically at 340nm and the enzyme activity was expressed as nmol
of NADPH produced/min/mg tissue protein.
The method of Krohne-Erich et al.20 was used to determine the activity of glutathione reductase (GR).The changes in absorbance was
recorded spectrophotometrically at 340nm.The specific activity of
the enzyme was expressed as unit/min/mg of protein.
Determination of the activities of pyruvate dehydrogenase (PDH)
and some of the Krebs cycle enzymes:
Method of Chretien et al. 21 with some modifications was adopted to
determine the activity of pyruvate dehydrogenase (PDH) by measuring the reduction of NAD+ to NADH at 340 nm. 50mM phosphate
buffer; pH - 7.4, 0.5 mM sodium pyruvate , 0.5mM NAD+ and optimum
amount of mitochondrial sample in 1ml assay mixture were used to
execute the assay. The enzyme activity was denoted as units/min/mg
of protein.
The activity of isocitrate dehydrogenase (ICDH) was determined by

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recording the reduction of NAD+ to NADH spectrophotometrically at
340 nm according to the method of Duncan and Fraenkel22. One ml
reaction mixture consisted of 50mM phosphate buffer;pH-7.4,0.5 mM
isocitrate,0.1 mM MnSO4,0.1mM NAD+ and sufficient amount of
sample containing mitochondria. The activity of this enzyme was
represented as unit/min/mg of protein.
Alpha-ketoglutarate dehydrogenase (a-KGDH) activity was determined by estimating the reduction of 0.35 mM NAD+ to NADH at 340
nm using 50 mM phosphate buffer, pH - 7.4, as the assay buffer, and
0.1 mM -ketoglutarate as the substrate according to the method
described by Duncan and Fraenkel22. The activity of this enzyme was
expressed as unit/min/mg of protein.
Succinate dehydrogenase (SDH) activity was measured according to
the method of Veeger et al. 23with some modifications12. Assay mixture consisted of 50mM phosphate buffer; pH - 7.4, 2%(w/v) BSA,
4mM succinate, 2.5mM K3Fe(CN)6 and optimum amount of sample
containing mitochondria as the source of enzyme. The enzyme activity was expressed as unit/min/mg of protein.
Aconitase activity was measured according to the method described
by Gardner et al24. Freshly isolated mitochondria were suspended in
0.5 ml of buffer containing 50 mM Tris-HCl (pH 7.4) and 0.6 mM
MnCl2 followed by the sonication for 2s. Aconitase activity was measured spectrophotometrically at 240 nm and at 250C in terms of absorbance of cis-aconitate gradually formed from externally added isocitrate (20 mM). One unit was defined as the amount of enzyme necessary to produce 1mol of cis-aconitate per minute (e240=3.6 mM1
cm-1). The enzyme activity was expressed as units/min/mg of protein.
Determination of the activities of mitochondrial respiratory chain
enzymes:
NADH-Cytochrome C oxidoreductase and cytochrome oxidase activities were measured according to the method described by Goyal
and Srivastava25. The activity of NADH-Cytochrome C oxidoreductase was measured spectrophotometrically by reduction of oxidized
cytochrome C at 565nm whereas activity of cytochrome oxidase was
also determined spectrophotometrically by oxidation of reduced cytochrome C at 550 nm.
Measurement of di-tyrosine fluorescence intensity in mitochondria:
Emission spectra of di-tyrosine, a product of tyrosine oxidation, were
recorded at 425nm (in the range 380 to 440 nm) (5 nm slit width) and
excitation wavelength was recorded at 325 nm (in the range 320 to 380
nm) (5 nm slit width) 26, 27.

Determination of tryptophan fluorescence in samples containing

mitochondria:
The fluorescence emission spectra (from 300 to 450 nm, 5 nm slit
width) of tryptophan were measured by excitation at 295 nm (2 nm slit
width) according to the procedure mentioned by Dousset et al.26.
Measurement of mitochondrial swelling:
To determine the mitochondrial swelling, the method of Halestrap
and Davidson was followed by recording the changes in the absorbance of the mitochondrial suspension spectrophotometrically at 520
nm. The changes in values of absorption at 520nm were evaluated to
determine the swelling of mitochondria28.
Determination of mitochondrial intactness by using Janus green B
stain:
Intactness of mitochondria was assessed by the procedure as followed by Dutta et al. 29. Mitochondrial smear was first prepared on a
slide. Following this, few drops of Janus green stain were put on the
slide and the slide was kept undisturbed in a moist chamber for 5
mins. After stipulated time period, the slide was rinsed carefully with
distilled water in such a manner that the stain was not washed away
but remained as diluted solution. The diluted stain with mitochondria
was covered with cover slip for confocal imaging. Confocal system
(BD Pathway 855, USA) was used for imaging.

Scanning electron microscopy:

The sample preparation for scanning electron microscopy was done
by the process as previously adopted by Dutta et al. 30with some
modifications. Images of the surfaces of the tissue and mitochondria
were captured by high definition camera attached with scanning electron microscope. (SEM; Zeiss Evo 18 model EDS 8100)
Histological studies:
A slice of the extirpated rat hearts and cerebrum portion of the brain
were fixed immediately in 10% formalin and embedded in paraffin
following routine procedure as used earlier by Mukherjee et al.31 and
Mitra et al.32. Left ventricular (LV) sections (5 m thick) and the sections of cerebrum of brain (5 m thick) were prepared and stained
with hematoxylin-eosin. The stained tissue sections were examined
under Leica microscope and the images were captured with a digital
camera attached to it.
Estimation of protein:
The protein content of all samples was determined by the method of
Lowry et al.33.

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16

Fig .1

14
12
10

**

8
6
4

0.8

0.7

**

0.5
0.4

Brain

0.3

Heart

0.2
0.1
0
CON

LPO level, PCO and GSH content of heart and cerebrum portion of
brain tissue were estimated to determine the efficacy of ISO in
generating oxidative stress in the mentioned organs. Figure 2A clearly
shows a significant rise (P<0.001 versus control) in LPO level of both
the tissues of the group treated with ISO. However, the LPO level of
the protected animals (pre-treated with melatonin 30 min prior to ISO
injection) was found significantly lower comparing to ISO group.
Figure 2B reveals ISO induced significant decrease in the GSH content
of the tissues which was almost completely protected from being
altered when pre-treated with melatonin. However, melatonin itself
has no significant effect on tissue GSH. Figure 2C demonstrates that
the PCO contents of both the tissues were significantly increased
following treatment of rats with ISO (P<0.001 versus control).This

Fig.2B

**

Heart

MEL

ISO

MEL+ISO

Groups

MEL + ISO

Fig.1. SGOT activity of different groups. CON: injected with

vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt.
ISO injected (s.c.)MEL +ISO: Pretreated with melatonin (40 mg/kg
b.wt.)i.p. and treated with ISO(25mg/kg b.wt)s.c.The values are expressed as meanSE; *P<0.001 compared to control values using
ANOVA. **P <0.001 compared to ISO treated values using ANOVA.

MEL + ISO

Brain

CON
ISO

ISO

45
40
35
30
25
20
15
10
5
0

0
MEL
Groups

MEL

Animal groups

2
CON

Fig.2A

0.6

Protein carbonyl level in tissues

nmoles of carbonyl/mg of
protein

Serum glutamate oxaloacetate

transaminase activity
IU/L

18

Lipid peroxidation level

nmoles TBARS/ mg of protein

RESULTS:
Figure 1 depicts that there is a remarkable increase in the activity of
SGOT in the serum of the rats treated with ISO which indicates the
myocardial tissue damage. The serum level of SGOT of ISO treated
rats reached the maximal value and showed significant change
compared to control (P<0.001 versus control).The pre-treatment of
rats with melatonin at a dose of 40mg/kg body weight checked the
rise in serum SGOT level.

elevated level of protein oxidation was significantly protected from

being increased when the rats were pre-treated with melatonin.
Melatonin alone have no significant effect on the level of PCO in
both the tissues.

GSH content
(nmole of GSH/mg of protein)

Statistical evaluation:
Every single experiment was repeated at least three times. Data are
represented as mean SE. One way analysis of variances (ANOVA)
followed by post hoc test (Tukeys HSD test) were done to determine
the significance of mean values of different parameters between the
different groups. Statistical tests were executed by using Microcal
Origin version 7.0.

Fig.2C

5
4

**

3
2

Brain
Heart

1
0
CON

MEL

ISO

MEL +
ISO

Groups
Fig.2.Protective effect of melatonin against ISO induced changes in
(A) lipid peroxidation(LPO) level (B) Reduced glutathione (GSH) and
(C)Protein carbonyl (PCO) content of heart and cerebrum portion of
brain tissues of rats of different groups. CON: injected with
vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt.
ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg
b.wt.)i.p. and treated with ISO(25mg/kg b.wt)s.c.The values are expressed as meanSE; *P<0.001 compared to control values using
ANOVA. **P<0.001 compared to ISO treated values using ANOVA.

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ISO

CON

MEL

ISO

MEL+ISO

Figure 4A clearly shows a significant rise (P< 0.001 versus control) in

LPO level in the mitochondria isolated from both the heart and brain
tissues of the group treated with ISO. However, the LPO level in the
mitochondria of the protected animals (pre-treated with melatonin 30
min prior to ISO injection) was found significantly lower comparing
to ISO group. Figure 4B reveals ISO induced significant decrease in
the GSH content of the mitochondria which were almost protected to
normal values when pre-treated with melatonin. Figure 4C demonstrate the PCO content of the mitochondrial fraction which was found
to be significantly increased following treatment of rats with ISO
(P< 0.001 versus control).This elevated level of protein oxidation was
significantly decreased when the rats were pre-treated with melatonin.
Lipid peroxidation level of
mitochondrial sample
nmoles of TBARS/mg of protein

60

Fig.4A
**

**

50
40

Fig.4B

Brain

30

Heart

20
10
0
CON

MEL

ISO

Groups

MEL+ISO

Fig.3.Protective effect of melatonin against ISO induced changes in

intactness of mitochondria separated from heart and cerebrum portion
of brain tissue. CON: injected with vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO:
Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with
ISO(25mg/kg b.wt),s.c.

20
18
16
14
12
10
8
6
4
2
0

70

Protein carbonyl level of

mitochondrial sample
nmole carbonyl / mg of protein

MEL

Heart (B)

CON

80

GSH content of mitochondrial

sample
nmoles/ mg of protein

Brain (A)

As depicted in Figure 3 (A and B), it is clear that ISO treatment significantly decreased the intactness of mitochondria isolated both from
the heart and cerebrum portion of the brain tissue. The mitochondrial
intactness was found to be normal in the groups where animals were
pre-treated with melatonin. However, melatonin alone has no effect
on the intactness of mitochondria obtained from both the tissues.

12

MEL +
ISO

10

Fig.4C

8
6

**

Brain
Heart

2
0
CON

MEL

ISO

MEL +
ISO

Groups
Fig.4.Protective effect of melatonin against ISO induced changes in
(A) Lipid Peroxidation(LPO) level (B) Reduced glutathione (GSH) and
(C)Protein carbonyl (PCO) content of mitochondrial fraction separated from heart and cerebrum portion of brain tissue of rats of different groups. CON: injected with vehicle,MEL: 40mg/kg b.wt melatonin
injected, i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/
kg b.wt),s.c.The values are expressed as meanSE; *P<0.001 compared
to control values using ANOVA. **P<0.001 compared to ISO treated
values using ANOVA.

Figure 5A demonstrates the activity of an important antioxidant enzyme Mn-SOD which is predominantly present in mitochondria and
its activity was found to be significantly increased by 1.97 fold in
brain and by 1.71 fold in heart of the groups treated with ISO compared to respective control. The activity of this enzyme in the group
pre-treated with melatonin was found to be significantly and almost
completely protected from being increased.

Brain
Heart

CON

MEL

ISO

Groups

MEL +
ISO

Figure 5(B and C) depicts the activities of other antioxidant enzymes

present in mitochondria of cardiac and cerebral tissue, such as GPx
and GR (though they are predominantly found in cytosol) which were
found to be significantly low in the groups treated with only ISO
while the enzyme activities were preserved to control level in the
animals which were pre-treated with melatonin.

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Mn-SOD activity
Units/min/mg of protein

30

Fig.5A

25

**

20

Brain

15

Heart

10
5
0
CON

MEL

Groups

ISO

MEL + ISO

3
Glutathione peroxidase activity of
mitochondrial sample
Units/min/mg of protein

2.5

**

Fig.5B
Brain

1.5

Heart

Figure 6(A-E) represents that PDH, aconitase, ICDH, -KGDH,and

SDH activities which were found to be inhibited in the mitochondria
of brain of ISO treated group respectively by 56.30%, 31.94%, 61.06%,
44.90% and 59.23% compared to control. Figure 6 (A-E) also represents that PDH, aconitase, ICDH, -KGDH and SDH activities of
mitochondria of heart which were found to be inhibited by 66.02%,
61.30%, 65.01%, 67.55% and 41.45% respectively, compared to their
control values. Pre-treatment of rats with melatonin elevated the activities of PDH, aconitase, ICDH, -KGDH and SDH, by 2.40 fold, 1.58
fold, 1.71 fold, 2.53 fold and 2.48 fold respectively, compared to ISO
treated group in brain and also 3.13 fold, 2.51 fold, 2.84 fold, 3.12 fold
, and 1.73 fold respectively, compared to cardiac mitochondria of the
ISO treated group.
70

Pyruvate dehydrogenase activity

Units/min/mg of protein

35

Fig.6A

50
40
30

Brain

20

Heart

10

0.5

0
CON

0
CON

MEL Groups ISO

MEL

ISO

MEL + ISO

Groups

MEL + ISO

** Fig. 5C

6
5
4
3

Brain

Heart

Aconitase activity
Units/min/mg of protein

7
6

**

5
4

Fig.6B
Brain

Heart

2
1
0
CON

MEL

ISO

MEL + ISO

Groups

0
CON

MEL

ISO

MEL + ISO

Groups
Fig.5.Protective effect of melatonin against ISO induced changes in
(A) Mn-SOD activity (B) Glutathione peroxidase activity and(C) Glutathione reductase activity of mitochondrial fraction separated from
heart and cerebrum portion of brain tissue of rats of different groups.
CON: injected with vehicle, MEL: 40mg/kg b.wt melatonin injected,
i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO: Pretreated with
melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/kg
b.wt),s.c.The values are expressed as mean SE;*P<0.001 compared to
control values using ANOVA. **P<0.001 compared to ISO treated values using ANOVA.

Isocitrate dehydrogenase activity

Units/mins/mg of protein

Glutathione reductase acitvity of

mitochondrial sample
Units/min/mg of protein

**

60

**

50
45
40
35
30
25
20
15
10
5
0

Fig.6C
Brain

CON

MEL

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Heart

MEL + ISO

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Fig.6D

50
40

30

Brain

20

Heart

10
0
CON

MEL

ISO

MEL +
ISO

Succinate Dehydrogenase activity

Units/min/mg of protein

Groups

300

Fig. 6E

200

150

Brain

100

Heart

50
0
CON

MEL

ISO

Fig.7A

2.5

**

2
1.5
1

MEL + ISO

Groups
Fig.6.Protective effect of melatonin against ISO induced changes in
Pyruvate dehydrogenase and some other Krebs cycle enzyme activities in mitochondrial fractions of heart and brain tissues (A) Succinate
dehydrogenase (SDH) activity (B)Isocitrate dehydrogenase (ICDH)
activity(C)Alpha ketoglutarate(-KGDH) activity (D)Pyruvate dehydrogenase (PDH) activity (E) Aconitase activity CON: injected with
vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt.
ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg
b.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.The values are expressed as meanSE; *P<0.001 compared to control values using
ANOVA. **P<0.001 compared to ISO treated values using ANOVA.

Figure.7. A and B depicts that ISO administration altered the normal

activities of respiratory chain enzymes. NADH Cytochrome C oxidoreductase and Cytochrome c oxidase activities were found to be
inhibited by70.78% and 55.56% respectively, in mitochondria of brain
and by 63.59% and 49% in mitochondria of heart tissue compared to
the respective control. Melatonin pre-treatment enhanced the NADH
Cytochrome C oxidoreductase and Cytochrome c oxidase activities

Brain

Heart

0.5
0
CON

MEL

ISO

Groups

**

250

by 3.42 fold and 2.4 fold in case of brain and by 2.89 fold and 2.14 fold
in case of heart compared to ISO treated group.

NADH Cytochrome c
oxido reductase
Units/min/mg of protein

**

Cytochroma C oxidase activity

Units/min/mg of protein

Alpha Ketogluatarate
dehydrogenase activity
Units/min/ mg of protein

60

MEL +
ISO

**

1.2
1
0.8
0.6
0.4
0.2
0

Fig.7B
*

CON

MEL
Groups

ISO

Brain
Heart

MEL +
ISO

Fig.7.Protective effect of melatonin against ISO induced changes in

the activities of respiratory enzymes.(A)NADH cytochrome C oxidoreductase activity(B)Cytochrome C oxidase activity.CON: injected
with vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg
b.wt. ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/
kg b.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.The values are
expressed as meanSE; *P<0.001 compared to control values using
ANOVA. **P<0.001 compared to ISO treated values using ANOVA.

Figure 8 depicts that ISO administration elevated the di-tyrosine fluorescence by 52.19% in mitochondria of brain (cerebral tissue) and by
2.09 fold in mitochondria of heart compared to respective control.
This indicates that ISO generates free radicals. But melatonin pretreatment of rats prevents di-tyrosine fluorescence values from being
increased. The fluorescence values recorded were less than 1.53 fold
in brain and 1.92 fold in heart compared to ISO-treated group. Di
tyrosine has been found to increase in oxidative stress due to exposure of tyrosine to oxygen free radicals, nitrogen dioxide, peroxynitrite,
and lipid hydroperoxides.

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Auroma Ghoshet al. / Journal of Pharmacy Research 2015,9(2),105-118

Fig.8

70
60

**

50
40

Brain

30

Heart

10

10

20

30

40

50

60

0
CON

MEL

ISO

MEL +
ISO

Figure.9 (A and B) reveals decreased fluorescence emission of tryptophan in mitochondria obtained from heart as well as cerebral cortical tissues in ISO treated group of rats but the fluorescence was
protected from being decreased when the melatonin was administered before the ISO injection. The decrease in fluorescence emission
indicates the destruction or modification of tryptophan. Here this
destruction or modification seems to occur due to oxidative stress.

A520 values

Fig.8.Protective effect of melatonin against ISO induced changes in

the di-tyrosine fluorescence intensity.CON: injected with vehicle,MEL:
40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt. ISO injected
s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. and
treated with ISO(25mg/kg b.wt),s.c.The values are expressed as
meanSE; *P<0.001 compared to control values using ANOVA.
**P<0.001 compared to ISO treated values using ANOVA.

-0.01
-0.02

CON
MEL

-0.03
-0.04

ISO
MEL + ISO

-0.05
-0.06

Fig.10 A

-0.07

10

20

Sec
30

40

50

60

0
CON
-0.05
-0.1

MEL
ISO
MEL + ISO

-0.15

450

Fluroscence intensity (AU)

Sec

20

Groups

400

Fig.9 A

350
300
200

MEL

150

ISO

100

MEL + ISO

50
300 310

320 330 340 350

360 370 380 390 400

410 420

Wavelength in nm
450

Fig.9B

400

-0.2
-0.25

CON

250

Fluroscence intensity (AU)

The increase in absorbance values at 520 nm is proportional to mitochondrial swelling. Figure 10 A and B depicts the enhancement of
mitochondrial swelling found in ISO treated animals compared to
control and positive control animals whereas melatonin pre-treatment of rats helped to bring the values to control level.

A520 valus

Di tyrosine level
Mean fluoroscence Intensity at
410nm (Arbitary unit)

80

Fig.10 B

Fig.10.Protective effect of melatonin against ISO induced changes in

the mitochondrial swelling in mitochondria isolated from rat heart
and brain.CON: injected with vehicle,MEL: 40mg/kg b.wt melatonin
injected, i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/
kg b.wt),s.c.

350
300

CON

250

MEL

200

ISO

150

MEL + ISO

100
50
0
300

310 320

330 340

350 360 370 380 390

400 410

420

Wavelength in nm

Fig.9.Protective effect of melatonin against ISO induced changes in

the fluorescence intensity of tryptophan. (A)Changes in heart (B)
Changes in brain.CON: injected with vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg b.wt. ISO injected s.c. MEL +ISO:
Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with
ISO(25mg/kg b.wt),s.c.

The observation of scanning electron microscopy represented in Figure 11 A and 12 A shows that the surface of both the tissues was
damaged following ISO treatment. The surface of the tissues became
fragile and kinky. However, in melatonin pre-treated group both the
tissues were significantly protected from deleterious effects of ISO.
Figure 11 B and 12 B shows that mitochondria isolated from heart and
brain respectively undergone massive changes when treated with
ISO. Perforated surface and convoluted membranes with blebs were
found in the mitochondria treated with ISO. Melatonin pre-treatment
of rats preserved the mitochondria which look close to the mitochondria obtained from control animals.

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Tissue ( A)

Auroma Ghoshet al. / Journal of Pharmacy Research 2015,9(2),105-118

MEL

ISO

MEL+ISO

Mitochondria (B)

CON

CON

MEL

ISO

MEL+ISO

Tissue ( A)

Fig.11.Representation of images of protective effect of melatonin against ISO induced changes in heart which were captured during Scanning
electron microscopy (X6000).(A)Tissue (B) Mitochondria.CON: injected with vehicle,MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/kg
b.wt. ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.

MEL

ISO

MEL+ISO

Mitochondria (B)

CON

CON

MEL

ISO

MEL+ISO

Fig.12.Representation of images of protective effect of melatonin against ISO induced changes in brain which were captured during Scanning
electron microscopy (X6000).(A)Tissue (B) Mitochondria.CON: injected with vehicle, MEL: 40mg/kg b.wt melatonin injected, i.p. ISO: 25mg/
kg b.wt. ISO injected s.c. MEL +ISO: Pretreated with melatonin (40 mg/kg b.wt.),i.p. and treated with ISO(25mg/kg b.wt),s.c.

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Auroma Ghoshet al. / Journal of Pharmacy Research 2015,9(2),105-118

Heart ( A)

The routine H & E stain of the tissues revealed that ISO at 25mg /kg
body wt, s.c. caused damage of cardiac tissue by creating myocardial
fibre necrosis but produced only sporadic edema in brain tissue.
However, when the rats were pre-treated with melatonin, these changes
in the cardiac and cerebral tissue morphology were found to be almost completely protected from being taken place.

MEL

ISO

MEL+ISO

Brain (B)

CON

CON

MEL

ISO

MEL+ISO

Fig. 13. (A) Changes of rat cardiac tissue morphology (H and E

stained, 40X magnification) (B) Changes in tissue morphology of
cerebrum portion of brain tissue. (H and E stained, 40X magnification)
DISCUSSION:
Many studies revealed that melatonin efficiently protects the heart
from myocardial injury resulted from oxidative stress8, 2. It is well
evident that oxidative stress generated by isoproterenol (ISO) not
only affects the tissue but also the mitochondria of myocytes34. A
study also showed that melatonin favorably changes the energy
metabolizing enzymes primarily altered by isoproterenol31. But to investigate the protection at mitochondrial level provided by melatonin
against oxidative stress in details we performed different biochemical
assays to determine the oxidative stress biomarkers, antioxidant enzyme status, activity of some of the Krebs cycle enzymes and also
some morphological and fluorescence studies in isolated mitochondria. The results clearly confirmed that melatonin not only beneficially changes the activity of mitochondrial enzymes but also preserves the mitochondrial integrity thus helping in the proper functioning of mitochondria and assisting in the recuperation from oxidative damages. Excess accumulation of reactive oxygen species (ROS)
in a system topples the antioxidant defense mechanisms thus oxidizing the essential biological marcomolecules like lipids, proteins, carbohydrates and DNA and it is well evident fact that these ROS along

with oxidized bio-molecules initiates inflammatory responses and

vascular or endothelial dysfunction leading to different degenerative
diseases like cardiovascular diseases, Alzheimers disease, diabetes
etc35, 36. After administration of isoproterenol in the living system the
oxidation of this compound generates quinones which upon further
oxidation form superoxide anion and hydrogen peroxide. The superoxide anion reduces tissue ferritin and thus produces hydroxyl radical and hydrogen peroxide. This hydroxyl radical is extremely reactive species and it starts the lipid peroxidation reaction2. One of the
aim of this study was to detect if isoproterenol itself or isoproterenol
induced myocardial injury can bring changes in the cerebral cortical
portion of the brain tissue and its mitochondria. We have found elevated level of LPO in the tissue and mitochondrial fraction of rat
cardiac and cerebrum portion of the brain tissue after administration
of isoproterenol. Melatonin pre-treatment was found to be effective
in decreasing the LPO level to normal in both the mitochondrial fraction and tissue through its free radical scavenging activity37.
Elevated level of reduced glutathione (GSH) strengthens the antioxidant capacity of a system thus preventing the generation of oxidative
stress38. Remarkable reduction in GSH content of both the tissue and
mitochondria occurred to elevate the level of oxidative stress generated by ISO. Melatonin pre-treatment elevated the GSH pool of both
the tissue and mitochondria by regulating the activity of two important enzymes such as glutathione peroxidase (GPx) and glutathione
reductase (GR) involved in the metabolism of reduced glutathione
(GSH) and oxidized glutathione (GSSG) 39.
The increased activity of Mn-SOD found in ISO treated group helps
in the alleviation of the oxidative stress 2 by quickly neutralizing superoxide anion. Pre-treatment with melatonin brought back the activity of Mn-SOD to normal level as melatonin detoxifies the superoxide
anion40.
The prime role of GPx is to protect the system from oxidative damage
by the free radicals41. The GR which is another important enzyme in
antioxidant defense system helps to regenerate GSH which is further
used to counteract the oxidative stress 42. The reduced activity of GPx
and GR were observed in the mitochondrial fraction of ISO treated
group. Melatonin pre-treatment was found to be effective in protecting the activities of these two enzymes as melatonin may spare these
enzymes by detoxifying the free radicals generated during oxidative
stress.
The reduced activity of Krebs cycle enzymes in ISO treated group
were found due to oxidative stress particularly hydrogen peroxide
stress which is directly formed upon the oxidation of ISO 31,43. It is

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seen that aconitase is the most sensitive enzyme in Krebs cycle as it
is readily inhibited by even the presence of small amount of hydrogen peroxide while inhibition of a ketoglutarate activity is seen in
higher concentration of hydrogen peroxide43. Decreased activities of
other TCA cycle enzymes were found when ISO was administered.
The main function of succinate dehydrogenase (SDH) enzyme is the
conversion of succinate to fumarate thereby transferring the electrons to electron transport chain. Thus, decreased activity of SDH
indicates limitation in electron flow and oxidative stress44. Decreased
activity of respiratory chain enzymes were also observed in both
brain and heart. Inhibited activity of Krebs cycle and respiratory
chain enzymes decrease the efficiency of electron transport chain
and facilitates the electron leakage thereby generating hydroxyl radical which further disrupts the mitochondrial membrane 31. Melatonin
pre-treatment protected the activities of these enzymes from being
altered thereby helping in the amelioration of oxidative stress induced changes due to ISO. The scanning electron microscopy of the
mitochondrial surface confirmed the disruption of mitochondrial surface in ISO treated group where as melatonin pre-treatment protected
the surface of this cell organelle from being damaged.
Mitochondria are the most important organelles present in the cell.
They are called the power house of the cell as energy production
occurred within the mitochondria. ATPs are generated by mitochondria when electrons are transported through the TCA cycle and respiratory chain complexes and thereby are very much susceptible to
oxidative stress.
The study clearly shows that melatonin is a highly efficient molecule
in curbing the oxidative damage 45not only in tissue level but also in
mitochondrial level. Some recent studies indicated that there may be
an association between ischemic heart disease and cognitive impairment46. From this study it can be concluded that either ISO directly
acts on brain like heart causing oxidative damage or ISO administration first causes myocardial infarction and ischemia which in turn
damages the brain. Further studies are essential to predict the direct
or indirect effect of ISO on the brain.
CONCLUSION:
It is clear from the study that melatonin protects both the mitochondria and the tissue of brain and heart against the oxidative damages
through its antioxidant mechanisms. So, the therapeutic use of melatonin may have beneficial effects on the people suffering from ischemic heart disease with brain damages.
ACKNOWLEDGEMENT:
This study was funded by a Major Research Project under CPEPA

scheme of UGC, Govt. of India, at University of Calcutta. AG, receiver

of UGC JRF fellowship, gratefully acknowledges UGC. MD is supported by Women Scientists Scheme A (WOS A), DST, Govt. of
India. Dr.AKG is the grantee of extended SRF under DST- PURSE
Program, Govt of India at University of Calcutta. Dr. AC is supported
from the funds available to her from Women Scientists Scheme A
(WOS A), DST, Govt. of India. DB is assisted by the funds available
to him from BD Bio-Sciences, Kolkata, India. We sincerely acknowledge and thank Sri Pratyush Sengupta for his technical help in respect of Scanning Electron Microscopy. We also acknowledge with
thanks the help provided to us by Center for Research in Nanoscience
and Nanotechnology (CRNN), University of Calcutta in respect of
using sophisticated instruments. Prof. DB thankfully acknowledges
the award of a Major Research Project under CPEPA Scheme of UGC,
Govt. of India, at University of Calcutta.
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Source of support: UGC, Govt. of India, Conflict of interest: None

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