Hindawi Publishing Corporation

BioMed Research International

Volume 2016, Article ID 6437641, 10 pages
http://dx.doi.org/10.1155/2016/6437641

Research Article
Sundarban Honey Confers Protection against
Isoproterenol-Induced Myocardial Infarction in Wistar Rats
Rizwana Afroz,1,2 E. M. Tanvir,1,3 Nurul Karim,1 Md. Sabir Hossain,1 Nadia Alam,4
Siew Hua Gan,4 and Md. Ibrahim Khalil1,4
1

Laboratory of Preventive and Integrative Biomedicine, Department of Biochemistry and Molecular Biology,
Jahangirnagar University, Savar, Dhaka 1342, Bangladesh
2
Department of Biochemistry, Primeasia University, Banani, Dhaka 1213, Bangladesh
3
Department of Biochemistry and Molecular Biology, Gonoshasthaya Samaj Vittik Medical College, Gono University,
Mirzanagar, Savar, Dhaka 1344, Bangladesh
4
Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
Correspondence should be addressed to Md. Ibrahim Khalil; drmikhalil@gmail.com
Received 16 March 2016; Revised 18 April 2016; Accepted 3 May 2016
Academic Editor: Tamer Mohamed
Copyright 2016 Rizwana Afroz et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The present study was designed to investigate the cardioprotective effects of Sundarban honey (SH) in rats with isoproterenol- (ISO-)
induced myocardial infarction. Adult male Wistar Albino rats were pretreated with Sundarban honey (5 g/kg) daily for a period of 6
weeks. After the treatment period, ISO (85 mg/kg) was subcutaneously injected into the rats at 24 h intervals for 2 days. ISO-induced
myocardial damage was indicated by increased serum cardiac specific troponin I levels and cardiac marker enzyme activities
including creatine kinase-MB, lactate dehydrogenase, aspartate transaminase, and alanine transaminase. Significant increases in
serum total cholesterol, triglycerides, and low-density lipoprotein-cholesterol levels were also observed, along with a reduction in
the serum high-density lipoprotein-cholesterol level. In addition to these diagnostic markers, the levels of lipid peroxide products
were significantly increased. The activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and
glutathione reductase were significantly decreased in the hearts after ISO-induced myocardial infarction. However, pretreatment
of ischemic rats with Sundarban honey brought the biochemical parameters to near normalcy, indicating the protective effect
of Sundarban honey against ISO-induced ischemia in rats. Histopathological findings of the heart tissues further confirmed the
biochemical findings, indicating that Sundarban honey confers protection against ISO-induced oxidative stress in the myocardium.

1. Introduction
Sundarban honey (SH) a wild multifloral honey produced by
Apis dorsata bees is one of the most renowned types of honey
from Bangladesh. Sundarban honey is collected from the
Sundarban region, the largest single block of tidal halophytic
mangrove forest in the world, located in the southern coastal
region of Bangladesh. Apis dorsata bees collect the nectar
from Sundarban plant species, for example, Khalsi [Aegiceras
corniculatum (L.) Blanco], Kakra [Bruguiera gymnorrhiza
(L.) Lam], Keora [Sonneratia apetala (B.) Ham], and Goran
[Ceriops decandra (G.) Ding Hou] [1]. Investigation on pharmacological benefits of SH revealed that it is one of the most

renowned types of honey from Bangladesh and conferred

protection against oxidative stress-induced liver and kidney
damage [2, 3].
Free radicals and reactive oxygen species have been
implicated in many diseases and have a deleterious effect on
cardiac function. Various experimental and clinical studies
have shown that enormous quantities of reactive oxygen
species including superoxide, H2 O2 , and hydroxyl radicals
are generated in the failing myocardium [4]. Therefore, therapeutic interventions that utilize antioxidants with free radical
scavenging activities have the potential to be used to combat
oxidative stress associated with various cardiovascular diseases, including myocardial infarction (MI). MI is a common

2
presentation of ischemic heart disease (IHD), a clinical syndrome arising from sudden and persistent curtailment of the
myocardial blood supply and resulting in myocardial necrosis [5, 6]. It is a complex phenomenon affecting the mechanical, electrical, structural, and biochemical properties of the
heart [7]. According to the World Health Organization, MI
is predicted to be the major cause of death worldwide by the
year 2020 [8].
Isoproterenol 4-[1-hydroxy-2-(isopropylamino)ethyl]benzene-1,2-diol hydrochloride (ISO) is a synthetic catecholamine and -adrenergic agonist documented to produce
severe stress in the myocardium and to result in MI if administered in supramaximal doses [9]. In the rat model, ISO
produces myocardial necrosis that leads to cardiac dysfunction, increased lipid peroxidation and increased levels of
myocardial lipids, and altered cardiac enzyme and antioxidant activities [4]. The proposed mechanisms to explain ISOinduced MI include generation of highly cytotoxic free radicals through the autoxidation of catecholamines [10]. These
free radicals may attack polyunsaturated fatty acids (PUFAs)
within the membranes, forming peroxyl radicals. The radicals can then attack adjacent fatty acids, causing a chain
reaction of lipid peroxidation (LPO). The lipid hydroperoxide end products are harmful and may contribute to
increased membrane permeability, leading to the development of cardiomyopathy [1012]. The pathophysiological and
morphological aberrations produced in hearts of the myocardial necrotic rat model are comparable with those that occur
in human MI.
In recent years, the prevention of cardiovascular disease
(CVD) has been associated with the consumption of fresh
fruits, vegetables, or plants rich in natural antioxidants,
because of superiority in terms of efficacy and safety when
compared with synthetic products [13]. Previous studies on
antioxidant potential of honeys revealed that Sundarban
honey contains the highest level of phenolics (688.50 mg gallic acid/kg), flavonoids (155.0 mg catechin/kg), ascorbic acid
(146.20 mg/kg), and protein (8.60 mg/g) content, as well as
the best free radical scavenging properties when compared
to other Bangladeshi honey samples [14]. In addition, the
presence of a number of phenolic acids including gallic, vanillic, and trans-cinnamic acids and pyrogallol and flavonoid
compounds including quercetin, naringin, and rutin has been
identified through high performance liquid chromatography
(HPLC) analysis [15]. Further insights into its capacity to
protect cells from the oxidative stress induced DNA damage
uncovered its medicinal importance related to degenerative
diseases such as cardiovascular diseases [15]. Epidemiological
studies on the cardioprotective effects of flavonoids suggest
that dietary intake of flavonoids has the potential to play
some preventive role in coronary heart diseases [16], while the
underlying cellular mechanism remains unknown. Therefore,
the present study was designed to investigate the cardioprotective effects of Sundarban honey to identify the possible
therapeutic efficacy on the activities of cardiac troponin I,
cardiac marker enzymes, the lipid profile, lipid peroxidation,
and antioxidant enzymes in rats with myocardial infarction
induced by supramaximal doses of ISO.

BioMed Research International

2. Materials and Methods

2.1. Experimental Animals. Adult male Wistar rats (170
190 g) were used in this study. Animals were bred and reared
in the animal housing facility of the Department of Biochemistry and Molecular Biology, Jahangirnagar University, in a
room with a constant temperature of 23 2 C and a humidity
ranging between 40% and 70%. The rats were housed in
plastic cages with hard-wood-chip bedding under a natural
12 h day-night cycle. The rats were provided with a standard
laboratory pellet diet and water ad libitum. The experiments
were conducted according to the ethical guidelines approved
by the Bangladesh Association for Laboratory Animal Science and the Biosafety, Biosecurity, and Ethical Committee
of Jahangirnagar University [Approval number BBEC, JU M
(2013.2.a)].
2.2. Drugs and Chemicals. The assay kit used to estimate cardiac troponin I (cTn I) was purchased from JAJ International,
Inc., San Diego, USA. Other assay kits for the measurement of
creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH),
aspartate transaminase (AST) and alanine transaminase
(ALT), total cholesterol (TC), triglycerides (TGs), and highdensity lipoprotein-cholesterol (HDL-C) were all purchased
from Stanbio Laboratory, USA. The assay kits for superoxide
dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GRx) were all purchased from Abnova
Corporation, Taiwan. ISO and 1,1,3,3-tetraethoxypropane
were purchased from Nacalai Tesque, Inc., Kyoto, Japan. All
of the chemicals and reagents used in this study were of
analytical grade.
2.3. Honey Sample. The multifloral honey sample was collected from Sundarban, Bangladesh, the largest mangrove
forest of the world, in February 2013.
2.4. Induction of Experimental MI. ISO was dissolved in normal saline and was subcutaneously injected into rats (85 mg/
kg) at 24 h intervals for 2 days to induce experimental MI.
The ISO dose was chosen based on a pilot study for ISO dose
fixation and on that used in previous studies [11, 17].
2.5. Experimental Design. After a one-week acclimation
period, animals were randomly divided into 4 groups (with
8 rats in each group) and were treated as follows:
Sham: animals received distilled water (2 mL/kg) for
6 weeks, followed by injection with normal saline
(1 mL) on the 43rd and 44th days.
SH + sham: animals received only SH (5 g/kg) for
6 weeks, followed by injection with normal saline
(1 mL) on the 43rd and 44th days.
SH + ISO: animals were orally pretreated with SH (5 g/
kg) by gastric gavage needle for 6 weeks, followed by
injection with ISO (85 mg/kg) on the 43rd and 44th
days.
ISO: animals received distilled water (2 mL/kg) for 6
weeks, followed by injection with ISO (85 mg/kg) on
the 43rd and 44th days.

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3
Rats (n = 32)

SH + sham
(n = 8)

Sham
(n = 8)

Administration
of distilled
water

SH + ISO
(n = 8)

Administration of SH
(5 g/kg b.w.)

ISO
(n = 8)

Administration
of distilled
water

For 6 weeks
Injected with normal saline
on 43rd and 44th day (24 h
interval)

Injected with ISO (85

mg/kg b.w.) on 43rd and
44th day (24 h interval)

Sacrifice of experimental animal

Collection of heart and blood samples for analysis

Figure 1: Schematic representation of experimental design of the study.

During the experimental period, the body weights of the

rats were regularly recorded and the doses were administered
accordingly. The dose of SH was also selected based on
the results of recent studies, in which the treated honey
sample (SH) and other honey samples showed strong hepatoprotective effects [2, 18]. Reduced locomotive activities
with increased water intake following the first ISO treatment
and worsened condition following the administration of
the second ISO dose were observed. All rats survived until
they were sacrificed. Forty-eight hours after the first dose
of ISO, all animals were sacrificed by decapitation. Blood
samples (3 mL) were collected, and serum was separated by
centrifugation. Immediately following blood collection, the
heart samples were separated from the surrounding tissues
and washed twice with ice-cold phosphate buffer saline (PBS).
The samples were homogenized in phosphate buffer (25 mM,
pH 7.4) to make approximately 10% w/v homogenates. The
homogenates were then centrifuged at 1700 rpm for 10 min,
and the supernatant was collected and stored at 20 C temperature until biochemical analysis. Some of the heart samples were stored in 10% formalin for histopathological examination (Figure 1).
2.6. Biochemical Analysis of Serum. An enzyme immunoassay kit was used for the determination of cardiac specific troponin I (cTn I) in serum samples using an ELISA microplate
reader (Digital and Analog System RS232, Das, Italy). Standard assay kits were used to determine the levels of CK-MB,
LDH, AST, ALT, TC, TGs, and HDL-C in serum samples
with a PD-303S Spectrophotometer (APEL, Japan). Serum

LDL-C levels were calculated based on a formula provided

by Friedewald et al. [19]:
LDL-C = TC [(

TGs
) + HDL-C] .
5

(1)

2.7. Biochemical Analysis of Heart Tissue. Malondialdehyde

(MDA) levels were assayed to detect lipid peroxidation
products in heart tissues. MDA, referred to as thiobarbituric
acid-reactive substance (TBARS), was measured at 532 nm
according to the method of Ohkawa et al. [20], and the levels
of TBARS are expressed as nmol of TBARS per mg of protein.
The heart tissue homogenates were recentrifuged at
12,000 rpm for 10 min at 4 C using Eppendorf centrifuge
5415D (Germany). Clean heart tissue supernatants were
obtained and used to estimate the activities of endogenous
antioxidative enzymes, including superoxide dismutase
(SOD), glutathione peroxidase (GPx), and glutathione reductase (GRx) using standard assay kits and a microplate reader
(Digital and Analog System RS232, Das, Italy). The levels of
SOD, GPx, and GRx are expressed as units/mg of protein,
nmol of NADPH oxidized/min/mg of protein, and nmol of
NADPH oxidized/min/mg of protein, respectively. The total
protein in heart tissue homogenates was estimated by the
method of Lowry et al. [21].
2.8. Histopathological Examination. After sacrifice, the heart
was rapidly dissected out and immediately washed with
saline, followed by fixation in 10% formalin. The fixed tissues
were embedded in paraffin and cut into serial sections
(5 m thick). Each section was stained with hematoxylin and

BioMed Research International

Table 1: The effects of SH on the body and heart weights of the rats.

Parameters
Initial body weight (g)
Final body weight (g)
Body weight gain (g)
Heart weight (g)

Sham
183.75 9.09a
219 3.54a
31.5 6.36a
0.85 0.01a

Treatment
SH + sham
SH + ISO
a
181.22 8.14
187.78 4.69a
a
206.11 5.44
220.22 8.39a
a
24.89 2.89
32.67 3.77a
a
0.84 0.01
1.07 0.02b

ISO
178.78 3.50a
217.83 3.06a
32.75 3.79a
1.14 0.01c

Percentage change
SH + sham
SH + ISO
ISO
1.38%
2.19%
2.70%
5.89%
0.56%
0.53%
20.98%
3.71%
3.96%
1.17%
25.88%
34.12%

Results are expressed as the mean SD, = 8. Values in the same row not sharing a common superscript (a, b, and c) differ significantly with each other at
< 0.05. Percentage change is calculated as 100 [(value of treatment value of sham)/value of sham].

2.9. Statistical Analysis. The results of all groups are shown as

the mean values standard deviation (SD). Data were analyzed using SPSS (Statistical Packages for Social Science,
version 20.0, IBM Corporation, New York, USA) and Microsoft Excel 2007 (Redmond, Washington, USA). Statistical
analyses of biochemical data were performed by Tukeys test;
< 0.05 was accepted as a statistically significant value.

0.9

0.8
0.7
cTn I (ng/mL)

eosin (H&E). Microscopic observation was performed using

a fluorescence microscope with normal spectra (Olympus
DP72, Tokyo, Japan). Photomicrographs were taken with a
digital camera (Olympus DP72, Tokyo, Japan) attached to the
microscope. The pathologist who performed the histopathological evaluation was blinded to the treatment assignments
of the different study groups.

0.6
0.5
0.4
0.3
0.2
0.1

b
a

Sham

SH + sham

0
SH + ISO

ISO

Figure 2: The effects of SH on serum cTn I levels. The bars represent

the mean SD ( = 8); bars with different letters are significantly
different at < 0.05.

There were no significant differences in the body weights

observed between the groups (Table 1). The heart weights
were significantly increased in ISO-administered rats compared with sham rats. In rats pretreated with SH prior to
ISO treatment, the heart weights were significantly reduced
compared with rats treated with ISO alone. No significant difference was observed in rats treated with SH alone compared
to the sham group.
Figure 2 shows the levels of serum troponin I (cTn I) in
normal and ISO-induced rats. Rats treated with ISO showed
considerably elevated ( < 0.05) serum cTn I levels compared
with normal control (sham) rats. ISO-induced rats pretreated
with SH daily for a period of 6 weeks showed a significant
( < 0.05) decrease in serum cTn I levels compared with rats
induced with ISO alone.
Figure 3 represents the effects of treatment with SH on
the activities of cardiac marker enzymes including CK-MB
and LDH AST and ALT in the serum of sham and ISOinduced rats. A marked increase in the activities of serum
cardiac enzymes was observed in ISO-induced myocardialischemic rats. However, there was a significant ( < 0.05)
decrease in the activities of CK-MB, LDH, AST, and ALT in
rats pretreated with SH before the ISO challenge.
The effects of SH treatment on the serum lipid profile (TC,
TGs, LDL-C, and HDL-C) of normal and ISO-treated rats are
listed in Table 2. Circulating levels of TC, TGs, and LDL-C
were significantly ( < 0.05) increased in ISO-treated rats,
whereas the levels of TC, TGs, and LDL-C were significantly

Enzyme activity (U/L)

3. Results
900
800
700
600
500
400
300
200
100
0

c
b
a a
a a b

CK-MB
Sham
SH + sham

a a
LDH

AST

a a b c
ALT

SH + ISO
ISO

Figure 3: The cardioprotective effects of SH on the activities of

cardiac marker enzymes. The bars represent the mean SD ( = 8);
bars with different letters are significantly different at < 0.05.

( < 0.05) decreased in the SH + ISO-treated group when

compared with the normal control group. A significant ( <
0.05) difference was also observed in serum HDL-C levels.
Increased levels of HDL-C were found in the SH + sham and
SH + ISO-treated groups compared with the sham and ISOtreated groups. Treatment with ISO significantly ( < 0.05)
reduced the levels of circulating HDL-C.
The effects of the oral administration of SH on LPO
levels of rat heart tissues were assessed in the present study
as a means to investigate whether SH has any potential to

BioMed Research International

Table 2: The antihyperlipidemic effects of SH on the serum lipid profiles.

Parameters
TC (mg/dL)
TGs (mg/dL)
LDL-C (mg/dL)
HDL-C (mg/dL)

Treatment
SH + sham
SH + ISO
a
47.54 0.80
57.74 3.13a
a
29.38 1.09
38.19 3.65ab
a
17.43 1.63
20.30 2.72a
b
31.89 0.23
30.78 1.47b

Sham
53.04 2.34a
38.13 2.07ab
19.69 1.62a
24.75 2.15a

ISO
72.48 2.41b
63.04 0.47c
40.36 1.58b
19.89 1.21c

SH + sham
10.37%
22.95%
11.48%
28.85%

Percentage change
SH + ISO
8.86%
0.16%
3.09%
24.36%

ISO
36.65%
65.33%
104.98%
19.63%

Results are expressed as the mean SD, = 8. Values in the same row not sharing a common superscript (a, b, and c) differ significantly with each other at
< 0.05. Percentage change is calculated as 100 [(value of treatment value of sham)/value of sham].

Table 3: The cardioprotective effects of SH on the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione
reductase (GRx) in the heart tissues of experimental animals.
Parameters

Treatment
SH + sham
SH + ISO

Sham

SOD (units/mg of
protein)
GPx (nmol NADPH
oxidized/min/mg of
protein)
GRx (nmol NADPH
oxidized/min/mg of
protein)

ISO

Percentage change
SH + sham
SH + ISO

ISO

1.71 0.14a

1.41 0.00a

0.92 0.01ab

0.02 0.00b

17.54%

46.19%

98.83%

3.18 0.00a

2.59 0.73a

2.31 0.06a

1.02 0.10b

18.55%

27.36%

67.92%

97.91 1.71a

99.79 3.83a

97.53 0.00a

81.69 1.56b

1.92%

0.39%

16.57%

LPO (nmol TBARS/mg of protein)

Results are expressed as the mean SD, = 8. Values in the same row not sharing a common superscript (a and b) differ significantly with each other at <
0.05. Percentage change is calculated as 100 [(value of treatment value of sham)/value of sham].

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Sham

SH + sham

SH + ISO

observation of widespread myocardial structural disorder,

coagulative necrosis, the separation of cardiac muscle fibers,
and infiltration of inflammatory cells. In rats pretreated with
SH, histopathological analysis revealed a decreased degree
of inflammatory cell infiltration and relatively well-preserved
cardiac muscle fiber morphology. Rats in the baseline group
(SH + sham) showed no changes in the histoarchitecture of
the heart tissue compared to normal control rats (Table 4).

4. Discussion
ISO

Figure 4: The cardioprotective effects of SH on cardiac LPO levels.

The bars represent the mean SD ( = 8); bars with different letters
are significantly different at < 0.05.

protect cardiac myocytes. Rats injected with ISO showed a

significant ( < 0.05) increase in the levels of LPO, while
prior treatment with SH significantly ( < 0.05) altered this
parameter. Figure 4 represents the LPO levels of the different
experimental group.
The effects of the oral administration of SH on antioxidant
enzyme levels in rat heart tissues were assessed in the present
study and the results are shown in Table 3.
Figure 5 shows histopathological photographs of the heart
tissues of experimental rats. Histopathological examination
of the myocardial tissue obtained from normal control rats
exhibited clear integrity of the myocardial membrane with
no observed inflammatory cell infiltration. Histopathological
findings confirmed the induction of MI by ISO based on the

Our study is the first to report the protective effects of

Sundarban honey against MI induced by ISO in rats. In the
present study, the heart weights increased significantly with
relatively unchanged body weights following ISO administration; this contributed to the increased heart weight to body
weight ratio. The increased heart weights might be attributed
to increased water content and edematous intramuscular
space [22]. However, pretreatment with SH maintained nearnormal heart weights.
The level of cTn I has been shown to be a highly
sensitive and specific marker of myocardial cell injury. These
contractile proteins are not normally found in the serum
and are released only after myocardial necrosis [23]. Elevated
troponin I levels predict the risk of both cardiac cell death
and subsequent infarction [24]. In our study, we observed
increased levels of serum cTn I in ISO-treated rats when
compared with normal control rats. The observed increase
in the levels of cTn I may be due to ISO-induced cardiac
damage. Rats challenged with ISO after pretreatment with SH
showed significantly decreased cTn I levels when compared

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Table 4: Semiquantitative scoring of the architectural changes evidenced by histopathological examination of rat myocardial tissues.
Treatment

Parameters

Sham

Membrane integrity
Separation of muscle fiber
Infiltration of inflammatory cells
Edematous intramuscular space

SH + sham

SH + ISO

++
+

ISO
+++
+++
+++
+++

Scoring was performed as follows: none (), mild (+), moderate (++), and severe (+++).

(a)

(c)

(b)

(d)

Figure 5: The effects of SH pretreatment on histopathological changes in the heart tissues of experimental rats. (a) Sham group: normal
control rat heart showing normal cardiac muscle fibers; (b) SH + sham group: SH pretreated rat heart showing normal muscle fibers without
any pathological changes; (c) SH + ISO group: SH pretreated rat myocardial tissues experiencing ISO challenge showing hyperplastic muscle
fibers and decreased degree of inflammatory cells; (d) ISO group: only ISO treated rat heart showing cardiac muscle fibers with muscle
separation (red arrows), edematous intramuscular space (green arrows), and inflammatory cell infiltration (yellow arrows). Magnification:
40x.

with rats treated with ISO alone. It is assumed that SH

may preserve the structural and functional integrity of the
contractile apparatus, which prevents cardiac damage and
leakage of troponins from the heart into the blood.
The myocardium contains high concentrations of diagnostic markers of MI and if metabolically damaged, releases
these contents into the extracellular fluids [22]. Of all of the
macromolecules that leak from the damaged tissue, enzymes
are the best markers of tissue damage because of their tissue
specificity and good catalytic activity. When myocardial cells
are damaged or destroyed due to a deficient oxygen supply
or the presence of high glucose concentrations, the cardiac
membrane becomes permeable and may rupture, which

results in the leakage of these enzymes [24]. The serum CKMB activity assay is an important and sensitive diagnostic tool
due to the high abundance of this enzyme in the myocardial
tissue and its virtual absence in most other tissues. CK-MB
isoenzyme activity is a useful early diagnostic index for MI or
any type of myocardial injury. Cytosolic enzymes including
CK-MB, LDH, AST, and ALT, which serve as diagnostic
markers, leak out from the damaged tissue into the blood
stream when the cell membrane becomes more permeable
or ruptures. The amount of these cellular enzymes in the
serum reflects alterations in the plasma membrane integrity
and/or permeability [25]. In the present study, rats exposed
to ISO showed significant elevations in the levels of all of

BioMed Research International

these marker enzymes in the serum. These results were in
line with previous reports and were indicative of ISO-induced
necrotic damage to the myocardium and leakiness of the
plasma membrane [22, 24, 26]. However, pretreatment with
SH resulted in lowered activities of all marker enzyme levels
in the serum, demonstrating that SH could contribute to the
maintenance of membrane integrity to restrict the leakage
of these enzymes. The phenolics such as gallic acid, transcinnamic acid, and vanillic acid and flavonoids including
quercetin, rutin, and naringin found in SH sample [15] are
speculated to confer protection by preserving the membrane
integrity [3] and restraining the leakage of these enzymes
from the myocardium.
Lipids play an important role in cardiovascular disease,
not only by contributing to the development of atherosclerosis but also by modifying the composition, structure, and
stability of the cellular membrane. High levels of circulating
cholesterol and its accumulation in the heart tissue have been
associated with cardiovascular damage [27]. Rats treated with
ISO showed a significant increase in the serum levels of TC,
TGs, and LDL-C, as previously reported [28]. Generally, the
mechanisms of actions of lipolytic hormones including ISO
on fat cells are believed to be mediated by the cAMP cascade,
in which lipolytic hormones activate adenylate cyclase and
thereby increase cAMP formation. Then, cAMP promotes
lipolytic activity by activating cAMP-dependent protein
kinase, which phosphorylates hormone-sensitive lipase [29]
and results in the hydrolysis of stored triacylglycerol, thereby
contributing to marked hyperlipidemia [30]. High levels of
LDL-C have been positively correlated with MI, while high
levels of HDL-C have a negative correlation. HDL-C inhibits
the uptake of LDL-C by the arterial walls and facilitates the
transport of cholesterol from peripheral tissues to the liver,
where it is catabolized and excreted from the body [31].
SH administration significantly restored these alterations,
thereby maintaining the normal fluidity and function of
the myocardial membrane. The mechanism by which SH
increases serum HDL-C and decreases TC, TGs, and LDLC levels is under investigation. The flavonoids such as rutin
have been identified in SH shown to inhibit platelet aggregation, anti-inflammatory effects, antioxidant, and reduction
of blood fat and cholesterol level. On the other hand, naringin has also been shown to exhibit cardioprotective effect
that might be via its cholesterol lowering and antihyperglycemic activities [32]. Alagwu et al. [33] postulated that
honey increases bile cholesterol excretion and lowers plasma
cholesterol levels. It is also plausible that honey reduces
plasma cholesterol through its antioxidant properties and by
enhancing the synthesis of HDL-C in the liver [34]. Polyphenols, particularly gallic acid and catechin, have been reported
to inhibit cholesterol esterase [35]. In general, pancreatic
cholesterol esterase plays an important role in hydrolyzing
dietary cholesterol esters, which liberates free cholesterol in
the lumen of the small intestine [36]. Therefore, the inhibition
of cholesterol esterase is expected to limit the absorbance of
dietary cholesterol, resulting in reduced cholesterol absorption. Moreover, polyphenols can also bind with bile acids to
increase their fecal excretion, which has been hypothesized as

7
a possible mechanism for the lowering of plasma cholesterol
levels by SH [35].
In recent years, research has increasingly focused on free
radicals that may modify biological molecules and result in
various pathological conditions [37]. Lipid peroxidation, a
type of oxidative deterioration of PUFAs, has been linked
with altered membrane structure and inactivation. In our
study, ISO treatment resulted in a significant increase in
the levels of lipid peroxidation products in the heart tissue.
Increased lipid peroxidation appears to be the initial stage
in making the tissue more susceptible to oxidative damage.
This may be responsible for the observed membrane damage
evidenced by elevated lipid peroxidation levels [24]. However,
pretreatment with SH showed significantly lowered levels
of lipid peroxides in ISO-induced rats. The antioxidant
nature of SH may hinder ROS production stimulated by ISO
administration and confer the protection to cardiac tissue.
The generation of ROS occurs due to the leakage of electrons from various systems into oxygen. Endogenous antioxidant enzymatic defense is a very important step in the neutralization of oxygen free radical-mediated tissue injury [38].
SOD, catalase, and GPx are the primary free radical scavenging enzymes involved in the first line of cellular defense
against oxidative injury, removing O2 and H2 O2 before they
can interact to form more reactive hydroxyl radicals [39, 40].
In this study, significantly lower SOD and GPx activities were
observed in the heart tissues of ISO-induced rats compared
with control rats. The observed decrease in the activities of
these enzymes may be due to increased generation of ROS
including hydrogen peroxide, superoxide, and hydroxyl radicals. This in turn leads to inactivation of these enzymes and
results in decreased removal of superoxide radicals, H2 O2 ,
and highly potent hydroxyl radicals [41, 42]. However, pretreatment with SH improved the scavenging activities of SOD
and GPx for superoxide and H2 O2 produced by ISO. GRx
is an antioxidant enzyme involved in the reduction of glutathione disulfide (GSSG) (an end product of the GPx reaction) to glutathione (GSH) [17]. In ISO-treated rats, a marked
reduction in GPx activity was observed, leading to reduced
availability of the substrate for GRx, thereby decreasing the
activity of GRx. However, oral pretreatment of ISO-treated
rats with SH restored the activity of GRx, thereby accelerating
the conversion of GSSG to GSH. The upregulation of the
activity or expression of Nrf2 is plausible; a transcription
factor released from its repressor (Keap1) under oxidative or
xenobiotic stress [43] is considered as possible mechanism
through which SH pretreatment restores antioxidant enzyme
functions as also suggested by Erejuwa et al. [44]. The
released Nrf2 binds to the antioxidant response element of
cytoprotective genes and induces their expression as well as
the subsequent expression of free radical scavenging enzymes
to neutralize and eliminate the cytotoxic oxidants [43].
The improvements in biochemical parameters reported
in the current study were more pronounced than the histopathological results. Nevertheless, histopathological examination of myocardial tissues in normal control rats illustrated
clear integrity of the myocardial cell membranes and no
inflammatory cell infiltration. Heart tissues from rats treated

BioMed Research International

Table 5: Comparison on percentage change for similar biomarkers between SH and TH pretreatment.

Parameters
cTn I (ng/mL)
CK-MB (U/L)
LPO (nmol TBARS/mg of protein)
SOD (units/mg of protein)
GPx (nmol NADPH oxidized/min/mg of protein)
TC
TGs

Sham

Tualang honey treatment

TH + ISO

0.06 0.01
94.44 3.05
9.25 0.68
1.46 0.05
2.86 0.06
45.55 2.49
46.31 3.68

0.18 0.05
158.03 7.59
16.84 1.06
0.19 0.02
1.86 0.20
56.77 3.70
53.37 3.91

ISO
0.69 0.05
242.39 7.30
22.42 1.09
0.16 0.01
1.07 0.19
76.52 4.84
72.88 3.28

Percentage change (%)

TH
SH
80.38
57.02
42.36
2.31
44.13
63.77
73.41

91.02
60.89
91.46
53.25
59.72
75.82
99.75

Results are expressed as the mean SD, = 10. Percentage of protection provided by each honey type is calculated as 100 [(value of ISO group value of
honey + ISO group)/(value of ISO group value of sham group)]. TH: Tualang honey and SH: Sundarban honey.

with ISO alone showed widespread myocardial structure disorder, coagulative necrosis, cardiac muscle fiber separation,
and inflammatory cell infiltration. However, the histopathological findings of the SH-pretreated myocardial infracted
hearts showed nearly normal morphologic cardiac muscle
characteristics and the absence of necrosis compared with
hearts from rats treated with ISO alone. The reduced inflammatory cell infiltration and cardiac muscle fiber architectural
damage further confirmed the cardioprotective effects of SH.
The findings on cardioprotective efficacy of SH were compared with Malaysian Tualang honey (TH) [41] for the
percentage changes in similar biological parameters (Table 5).
SH showed better effects compared to TH when MI was
induced by ISO at 85 mg/kg body weight.
Overall, the probable mechanism by which SH exhibited
its protective and ameliorative effects against ISO-induced
myocardial damage is via improved antioxidative status and
lowered plasma cholesterol level. Further studies should be
conducted to clarify the exact mechanism of the cardioprotective effect of SH.

5. Conclusion
Pretreatment with SH significantly altered nearly all biochemical parameters associated with ISO-induced myocardial injury. These findings were confirmed by histopathological examination of normal and infracted rat hearts. This
finding may provide scientific support to understand the beneficial effects of SH on cardioprotection against myocardial
injury, in which oxidative stress has long been known to contribute to the pathogenesis.

Competing Interests
The authors declare that there is no conflict of interests
regarding the publication of this paper.

Acknowledgments
This research is partly supported by the Bangladesh Medical
Research Council (BMRC) Grant 2012-2013 and partly by
TWAS Research Grant no. 12-237 RG/PHA/AS C; UNESCO

FR: 3240270864 and TWAS Research Grant no. 14-385 RG/

PHA/AS C; UNESCO FR: 3240283438.

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