Research Article Sundarban Honey Confers Protection ...downloads.hindawi.com/journals/bmri/2016/6437641.pdf · Research Article Sundarban Honey Confers Protection against Isoproterenol-Induced

Research ArticleSundarban Honey Confers Protection againstIsoproterenol-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

1Laboratory of Preventive and Integrative Biomedicine, Department of Biochemistry and Molecular Biology,Jahangirnagar University, Savar, Dhaka 1342, Bangladesh2Department of Biochemistry, Primeasia University, Banani, Dhaka 1213, Bangladesh3Department of Biochemistry and Molecular Biology, Gonoshasthaya Samaj Vittik Medical College, Gono University,Mirzanagar, Savar, Dhaka 1344, Bangladesh4Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

Correspondence should be addressed to Md. Ibrahim Khalil; [email protected]

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.

Thepresent studywas designed to investigate the cardioprotective effects of Sundarbanhoney (SH) in ratswith isoproterenol- (ISO-)inducedmyocardial infarction. Adult maleWistar Albino rats were pretreated with Sundarban honey (5 g/kg) daily for a period of 6weeks. After the treatment period, ISO (85mg/kg) was subcutaneously injected into the rats at 24 h intervals for 2 days. ISO-inducedmyocardial damage was indicated by increased serum cardiac specific troponin I levels and cardiac marker enzyme activitiesincluding creatine kinase-MB, lactate dehydrogenase, aspartate transaminase, and alanine transaminase. Significant increases inserum total cholesterol, triglycerides, and low-density lipoprotein-cholesterol levels were also observed, along with a reduction inthe serum high-density lipoprotein-cholesterol level. In addition to these diagnostic markers, the levels of lipid peroxide productswere significantly increased. The activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, andglutathione reductase were significantly decreased in the hearts after ISO-induced myocardial infarction. However, pretreatmentof ischemic rats with Sundarban honey brought the biochemical parameters to near normalcy, indicating the protective effectof Sundarban honey against ISO-induced ischemia in rats. Histopathological findings of the heart tissues further confirmed thebiochemical findings, indicating that Sundarban honey confers protection against ISO-induced oxidative stress in themyocardium.

1. Introduction

Sundarban honey (SH) a wild multifloral honey produced byApis dorsata bees is one of the most renowned types of honeyfrom Bangladesh. Sundarban honey is collected from theSundarban region, the largest single block of tidal halophyticmangrove forest in the world, located in the southern coastalregion of Bangladesh. Apis dorsata bees collect the nectarfrom Sundarban plant species, for example, Khalsi [Aegicerascorniculatum (L.) Blanco], Kakra [Bruguiera gymnorrhiza(L.) Lam], Keora [Sonneratia apetala (B.) Ham], and Goran[Ceriops decandra (G.) Ding Hou] [1]. Investigation on phar-macological benefits of SH revealed that it is one of the most

renowned types of honey from Bangladesh and conferredprotection against oxidative stress-induced liver and kidneydamage [2, 3].

Free radicals and reactive oxygen species have beenimplicated in many diseases and have a deleterious effect oncardiac function. Various experimental and clinical studieshave shown that enormous quantities of reactive oxygenspecies including superoxide, H

2O2, and hydroxyl radicals

are generated in the failingmyocardium [4].Therefore, thera-peutic interventions that utilize antioxidants with free radicalscavenging activities have the potential to be used to combatoxidative stress associated with various cardiovascular dis-eases, including myocardial infarction (MI). MI is a common

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016, Article ID 6437641, 10 pageshttp://dx.doi.org/10.1155/2016/6437641

2 BioMed Research International

presentation of ischemic heart disease (IHD), a clinical syn-drome arising from sudden and persistent curtailment of themyocardial blood supply and resulting in myocardial necro-sis [5, 6]. It is a complex phenomenon affecting the mechan-ical, electrical, structural, and biochemical properties of theheart [7]. According to the World Health Organization, MIis predicted to be the major cause of death worldwide by theyear 2020 [8].

Isoproterenol 4-[1-hydroxy-2-(isopropylamino)ethyl]ben-zene-1,2-diol hydrochloride (ISO) is a synthetic catechola-mine and 𝛽-adrenergic agonist documented to producesevere stress in the myocardium and to result inMI if admin-istered in supramaximal doses [9]. In the rat model, ISOproduces myocardial necrosis that leads to cardiac dysfunc-tion, increased lipid peroxidation and increased levels ofmyocardial lipids, and altered cardiac enzyme and antioxi-dant activities [4].The proposedmechanisms to explain ISO-induced MI include generation of highly cytotoxic free rad-icals through the autoxidation of catecholamines [10]. Thesefree radicals may attack polyunsaturated fatty acids (PUFAs)within the membranes, forming peroxyl radicals. The rad-icals can then attack adjacent fatty acids, causing a chainreaction of lipid peroxidation (LPO). The lipid hydroper-oxide end products are harmful and may contribute toincreased membrane permeability, leading to the develop-ment of cardiomyopathy [10–12].The pathophysiological andmorphological aberrations produced in hearts of themyocar-dial necrotic rat model are comparable with those that occurin human MI.

In recent years, the prevention of cardiovascular disease(CVD) has been associated with the consumption of freshfruits, vegetables, or plants rich in natural antioxidants,because of superiority in terms of efficacy and safety whencompared with synthetic products [13]. Previous studies onantioxidant potential of honeys revealed that Sundarbanhoney contains the highest level of phenolics (688.50mg gal-lic acid/kg), flavonoids (155.0mg catechin/kg), ascorbic acid(146.20mg/kg), and protein (8.60mg/g) content, as well asthe best free radical scavenging properties when comparedto other Bangladeshi honey samples [14]. In addition, thepresence of a number of phenolic acids including gallic, vanil-lic, and trans-cinnamic acids and pyrogallol and flavonoidcompounds including quercetin, naringin, and rutin has beenidentified through high performance liquid chromatography(HPLC) analysis [15]. Further insights into its capacity toprotect cells from the oxidative stress induced DNA damageuncovered its medicinal importance related to degenerativediseases such as cardiovascular diseases [15]. Epidemiologicalstudies on the cardioprotective effects of flavonoids suggestthat dietary intake of flavonoids has the potential to playsomepreventive role in coronary heart diseases [16], while theunderlying cellularmechanism remains unknown.Therefore,the present study was designed to investigate the cardiopro-tective effects of Sundarban honey to identify the possibletherapeutic efficacy on the activities of cardiac troponin I,cardiac marker enzymes, the lipid profile, lipid peroxidation,and antioxidant enzymes in rats with myocardial infarctioninduced by supramaximal doses of ISO.

2. Materials and Methods

2.1. Experimental Animals. Adult male Wistar rats (170–190 g) were used in this study. Animals were bred and rearedin the animal housing facility of the Department of Biochem-istry and Molecular Biology, Jahangirnagar University, in aroomwith a constant temperature of 23 ± 2∘C and a humidityranging between 40% and 70%. The rats were housed inplastic cages with hard-wood-chip bedding under a natural12 h day-night cycle. The rats were provided with a standardlaboratory pellet diet and water ad libitum. The experimentswere conducted according to the ethical guidelines approvedby the Bangladesh Association for Laboratory Animal Sci-ence and the Biosafety, Biosecurity, and Ethical Committeeof Jahangirnagar University [Approval number BBEC, JU M(2013.2.a)].

2.2. Drugs and Chemicals. The assay kit used to estimate car-diac troponin I (cTn I) was purchased from JAJ International,Inc., SanDiego, USA.Other assay kits for themeasurement ofcreatine kinase-MB (CK-MB), lactate dehydrogenase (LDH),aspartate transaminase (AST) and alanine transaminase(ALT), total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein-cholesterol (HDL-C) were all purchasedfrom Stanbio Laboratory, USA. The assay kits for superoxidedismutase (SOD), glutathione peroxidase (GPx), and glu-tathione reductase (GRx) were all purchased from AbnovaCorporation, Taiwan. ISO and 1,1,3,3-tetraethoxypropanewere purchased from Nacalai Tesque, Inc., Kyoto, Japan. Allof the chemicals and reagents used in this study were ofanalytical grade.

2.3. Honey Sample. The multifloral honey sample was col-lected from Sundarban, Bangladesh, the largest mangroveforest of the world, in February 2013.

2.4. Induction of Experimental MI. ISO was dissolved in nor-mal saline and was subcutaneously injected into rats (85mg/kg) at 24 h intervals for 2 days to induce experimental MI.The ISO dose was chosen based on a pilot study for ISO dosefixation and on that used in previous studies [11, 17].

2.5. Experimental Design. After a one-week acclimationperiod, animals were randomly divided into 4 groups (with8 rats in each group) and were treated as follows:

Sham: animals received distilled water (2mL/kg) for6 weeks, followed by injection with normal saline(1mL) on the 43rd and 44th days.SH + sham: animals received only SH (5 g/kg) for6 weeks, followed by injection with normal saline(1mL) on the 43rd and 44th days.SH+ ISO: animalswere orally pretreatedwith SH (5 g/kg) by gastric gavage needle for 6 weeks, followed byinjection with ISO (85mg/kg) on the 43rd and 44thdays.ISO: animals received distilled water (2mL/kg) for 6weeks, followed by injection with ISO (85mg/kg) onthe 43rd and 44th days.

BioMed Research International 3

Administration of distilled

water

Administration of distilled

water

For 6 weeks

Sacrifice of experimental animal

Collection of heart and blood samples for analysis

Rats (n = 32)

Sham (n = 8)

SH + sham (n = 8)

SH + ISO (n = 8)

ISO(n = 8)

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

Injected with normal saline

interval)3rd and 44th day (24h

Injected with ISO (85 mg/kg b.w.) on 43rd and44th day (24h interval)

on 4

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

During the experimental period, the body weights of therats were regularly recorded and the doses were administeredaccordingly. The dose of SH was also selected based onthe results of recent studies, in which the treated honeysample (SH) and other honey samples showed strong hep-atoprotective effects [2, 18]. Reduced locomotive activitieswith increased water intake following the first ISO treatmentand worsened condition following the administration ofthe second ISO dose were observed. All rats survived untilthey were sacrificed. Forty-eight hours after the first doseof ISO, all animals were sacrificed by decapitation. Bloodsamples (3mL) were collected, and serum was separated bycentrifugation. Immediately following blood collection, theheart samples were separated from the surrounding tissuesandwashed twicewith ice-cold phosphate buffer saline (PBS).The samples were homogenized in phosphate buffer (25mM,pH 7.4) to make approximately 10%w/v homogenates. Thehomogenates were then centrifuged at 1700 rpm for 10min,and the supernatant was collected and stored at −20∘C tem-perature until biochemical analysis. Some of the heart sam-ples were stored in 10% formalin for histopathological exam-ination (Figure 1).

2.6. Biochemical Analysis of Serum. An enzyme immunoas-say kit was used for the determination of cardiac specific trop-onin I (cTn I) in serum samples using an ELISA microplatereader (Digital and Analog System RS232, Das, Italy). Stan-dard assay kits were used to determine the levels of CK-MB,LDH, AST, ALT, TC, TGs, and HDL-C in serum sampleswith a PD-303S Spectrophotometer (APEL, Japan). Serum

LDL-C levels were calculated based on a formula providedby Friedewald et al. [19]:

LDL-C = TC − [(TGs5) +HDL-C] . (1)

2.7. Biochemical Analysis of Heart Tissue. Malondialdehyde(MDA) levels were assayed to detect lipid peroxidationproducts in heart tissues. MDA, referred to as thiobarbituricacid-reactive substance (TBARS), was measured at 532 nmaccording to the method of Ohkawa et al. [20], and the levelsof TBARS are expressed as nmol of TBARS permg of protein.

The heart tissue homogenates were recentrifuged at12,000 rpm for 10min at 4∘C using Eppendorf centrifuge5415D (Germany). Clean heart tissue supernatants wereobtained and used to estimate the activities of endogenousantioxidative enzymes, including superoxide dismutase(SOD), glutathione peroxidase (GPx), and glutathione reduc-tase (GRx) using standard assay kits and a microplate reader(Digital and Analog System RS232, Das, Italy). The levels ofSOD, GPx, and GRx are expressed as units/mg of protein,nmol of NADPH oxidized/min/mg of protein, and nmol ofNADPH oxidized/min/mg of protein, respectively. The totalprotein in heart tissue homogenates was estimated by themethod of Lowry et al. [21].

2.8. Histopathological Examination. After sacrifice, the heartwas rapidly dissected out and immediately washed withsaline, followed by fixation in 10% formalin. The fixed tissueswere embedded in paraffin and cut into serial sections(5 𝜇m thick). Each section was stained with hematoxylin and


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

Parameters Treatment Percentage changeSham SH + sham SH + ISO ISO SH + sham SH + ISO ISO

Initial body weight (g) 183.75 ± 9.09a 181.22 ± 8.14a 187.78 ± 4.69a 178.78 ± 3.50a −1.38% 2.19% −2.70%Final body weight (g) 219 ± 3.54a 206.11 ± 5.44a 220.22 ± 8.39a 217.83 ± 3.06a −5.89% 0.56% −0.53%Body weight gain (g) 31.5 ± 6.36a 24.89 ± 2.89a 32.67 ± 3.77a 32.75 ± 3.79a −20.98% 3.71% 3.96%Heart weight (g) 0.85 ± 0.01a 0.84 ± 0.01a 1.07 ± 0.02b 1.14 ± 0.01c −1.17% 25.88% 34.12%Results are expressed as the mean ± SD, 𝑛 = 8. Values in the same row not sharing a common superscript (a, b, and c) differ significantly with each other at𝑝 < 0.05. Percentage change is calculated as 100 × [(value of treatment − value of sham)/value of sham].

eosin (H&E). Microscopic observation was performed usinga fluorescence microscope with normal spectra (OlympusDP72, Tokyo, Japan). Photomicrographs were taken with adigital camera (Olympus DP72, Tokyo, Japan) attached to themicroscope. The pathologist who performed the histopatho-logical evaluation was blinded to the treatment assignmentsof the different study groups.

2.9. Statistical Analysis. The results of all groups are shown asthe mean values ± standard deviation (SD). Data were ana-lyzed using SPSS (Statistical Packages for Social Science,version 20.0, IBM Corporation, New York, USA) and Mic-rosoft Excel 2007 (Redmond, Washington, USA). Statisticalanalyses of biochemical data were performed by Tukey’s test;𝑝 < 0.05 was accepted as a statistically significant value.

3. Results

There were no significant differences in the body weightsobserved between the groups (Table 1). The heart weightswere significantly increased in ISO-administered rats com-pared with sham rats. In rats pretreated with SH prior toISO treatment, the heart weights were significantly reducedcomparedwith rats treatedwith ISO alone. No significant dif-ference was observed in rats treated with SH alone comparedto the sham group.

Figure 2 shows the levels of serum troponin I (cTn I) innormal and ISO-induced rats. Rats treated with ISO showedconsiderably elevated (𝑝 < 0.05) serum cTn I levels comparedwith normal control (sham) rats. ISO-induced rats pretreatedwith SH daily for a period of 6 weeks showed a significant(𝑝 < 0.05) decrease in serum cTn I levels compared with ratsinduced with ISO alone.

Figure 3 represents the effects of treatment with SH onthe activities of cardiac marker enzymes including CK-MBand LDH AST and ALT in the serum of sham and ISO-induced rats. A marked increase in the activities of serumcardiac enzymes was observed in ISO-induced myocardial-ischemic rats. However, there was a significant (𝑝 < 0.05)decrease in the activities of CK-MB, LDH, AST, and ALT inrats pretreated with SH before the ISO challenge.

The effects of SH treatment on the serum lipid profile (TC,TGs, LDL-C, andHDL-C) of normal and ISO-treated rats arelisted in Table 2. Circulating levels of TC, TGs, and LDL-Cwere significantly (𝑝 < 0.05) increased in ISO-treated rats,whereas the levels of TC, TGs, and LDL-C were significantly

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Sham SH + sham SH + ISO ISO

aab

c

cTn

I (ng

/mL)

Figure 2:The effects of SH on serum cTn I levels.The bars representthe mean ± SD (𝑛 = 8); bars with different letters are significantlydifferent at 𝑝 < 0.05.

0100200300400500600700800900

CK-MB LDH AST ALT

Enzy

me a

ctiv

ity (U

/L)

ShamSH + sham

SH + ISOISO

a

a

aaa

aa

a

bb

b

b

c

c

c

c

Figure 3: The cardioprotective effects of SH on the activities ofcardiac marker enzymes. The bars represent the mean ± SD (𝑛 = 8);bars with different letters are significantly different at 𝑝 < 0.05.

(𝑝 < 0.05) decreased in the SH + ISO-treated group whencompared with the normal control group. A significant (𝑝 <0.05) difference was also observed in serum HDL-C levels.Increased levels of HDL-C were found in the SH + sham andSH + ISO-treated groups compared with the sham and ISO-treated groups. Treatment with ISO significantly (𝑝 < 0.05)reduced the levels of circulating HDL-C.

The effects of the oral administration of SH on LPOlevels of rat heart tissues were assessed in the present studyas a means to investigate whether SH has any potential to


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


TC (mg/dL) 53.04 ± 2.34a 47.54 ± 0.80a 57.74 ± 3.13a 72.48 ± 2.41b −10.37% 8.86% 36.65%TGs (mg/dL) 38.13 ± 2.07ab 29.38 ± 1.09a 38.19 ± 3.65ab 63.04 ± 0.47c −22.95% 0.16% 65.33%LDL-C (mg/dL) 19.69 ± 1.62a 17.43 ± 1.63a 20.30 ± 2.72a 40.36 ± 1.58b −11.48% 3.09% 104.98%HDL-C (mg/dL) 24.75 ± 2.15a 31.89 ± 0.23b 30.78 ± 1.47b 19.89 ± 1.21c 28.85% 24.36% −19.63%Results are expressed as the mean ± SD, 𝑛 = 8. Values in the same row not sharing a common superscript (a, b, and c) differ significantly with each other at𝑝 < 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 glutathionereductase (GRx) in the heart tissues of experimental animals.


SOD (units/mg ofprotein) 1.71 ± 0.14a 1.41 ± 0.00a 0.92 ± 0.01ab 0.02 ± 0.00b −17.54% −46.19% −98.83%

GPx (nmol NADPHoxidized/min/mg ofprotein)

3.18 ± 0.00a 2.59 ± 0.73a 2.31 ± 0.06a 1.02 ± 0.10b −18.55% −27.36% −67.92%

GRx (nmol NADPHoxidized/min/mg ofprotein)

97.91 ± 1.71a 99.79 ± 3.83a 97.53 ± 0.00a 81.69 ± 1.56b 1.92% −0.39% −16.57%

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

00.20.40.60.8

11.21.41.61.8

2

Sham SH + sham SH + ISO ISO

a a b

c

LPO

(nm

ol T

BARS

/mg

of p

rote

in)

Figure 4: The cardioprotective effects of SH on cardiac LPO levels.The bars represent the mean ± SD (𝑛 = 8); bars with different lettersare significantly different at 𝑝 < 0.05.

protect cardiac myocytes. Rats injected with ISO showed asignificant (𝑝 < 0.05) increase in the levels of LPO, whileprior treatment with SH significantly (𝑝 < 0.05) altered thisparameter. Figure 4 represents the LPO levels of the differentexperimental group.

The effects of the oral administration of SHon antioxidantenzyme levels in rat heart tissues were assessed in the presentstudy and the results are shown in Table 3.

Figure 5 shows histopathological photographs of the hearttissues of experimental rats. Histopathological examinationof the myocardial tissue obtained from normal control ratsexhibited clear integrity of the myocardial membrane withno observed inflammatory cell infiltration. Histopathologicalfindings confirmed the induction of MI by ISO based on the

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

4. Discussion

Our study is the first to report the protective effects ofSundarban honey against MI induced by ISO in rats. In thepresent study, the heart weights increased significantly withrelatively unchanged bodyweights following ISO administra-tion; this contributed to the increased heart weight to bodyweight ratio. The increased heart weights might be attributedto increased water content and edematous intramuscularspace [22]. However, pretreatment with SHmaintained near-normal heart weights.

The level of cTn I has been shown to be a highlysensitive and specific marker of myocardial cell injury. Thesecontractile proteins are not normally found in the serumand are released only after myocardial necrosis [23]. Elevatedtroponin I levels predict the risk of both cardiac cell deathand subsequent infarction [24]. In our study, we observedincreased levels of serum cTn I in ISO-treated rats whencompared with normal control rats. The observed increasein the levels of cTn I may be due to ISO-induced cardiacdamage. Rats challengedwith ISO after pretreatment with SHshowed significantly decreased cTn I levels when compared


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

Parameters TreatmentSham SH + sham SH + ISO ISO

Membrane integrity − − − +++Separation of muscle fiber − − − +++Infiltration of inflammatory cells − − ++ +++Edematous intramuscular space − − + +++Scoring was performed as follows: none (−), mild (+), moderate (++), and severe (+++).

(a) (b)

(c) (d)

Figure 5: The effects of SH pretreatment on histopathological changes in the heart tissues of experimental rats. (a) Sham group: normalcontrol rat heart showing normal cardiac muscle fibers; (b) SH + sham group: SH pretreated rat heart showing normal muscle fibers withoutany pathological changes; (c) SH + ISO group: SH pretreated rat myocardial tissues experiencing ISO challenge showing hyperplastic musclefibers and decreased degree of inflammatory cells; (d) ISO group: only ISO treated rat heart showing cardiac muscle fibers with muscleseparation (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 SHmay preserve the structural and functional integrity of thecontractile apparatus, which prevents cardiac damage andleakage of troponins from the heart into the blood.

The myocardium contains high concentrations of diag-nostic markers of MI and if metabolically damaged, releasesthese contents into the extracellular fluids [22]. Of all of themacromolecules that leak from the damaged tissue, enzymesare the best markers of tissue damage because of their tissuespecificity and good catalytic activity. When myocardial cellsare damaged or destroyed due to a deficient oxygen supplyor the presence of high glucose concentrations, the cardiacmembrane becomes permeable and may rupture, which

results in the leakage of these enzymes [24]. The serum CK-MBactivity assay is an important and sensitive diagnostic tooldue to the high abundance of this enzyme in the myocardialtissue and its virtual absence in most other tissues. CK-MBisoenzyme activity is a useful early diagnostic index forMI orany type of myocardial injury. Cytosolic enzymes includingCK-MB, LDH, AST, and ALT, which serve as diagnosticmarkers, leak out from the damaged tissue into the bloodstream when the cell membrane becomes more permeableor ruptures. The amount of these cellular enzymes in theserum reflects alterations in the plasma membrane integrityand/or permeability [25]. In the present study, rats exposedto ISO showed significant elevations in the levels of all of


these marker enzymes in the serum. These results were inlinewith previous reports andwere indicative of ISO-inducednecrotic damage to the myocardium and leakiness of theplasma membrane [22, 24, 26]. However, pretreatment withSH resulted in lowered activities of all marker enzyme levelsin the serum, demonstrating that SH could contribute to themaintenance of membrane integrity to restrict the leakageof these enzymes. The phenolics such as gallic acid, trans-cinnamic acid, and vanillic acid and flavonoids includingquercetin, rutin, and naringin found in SH sample [15] arespeculated to confer protection by preserving the membraneintegrity [3] and restraining the leakage of these enzymesfrom the myocardium.

Lipids play an important role in cardiovascular disease,not only by contributing to the development of atheroscle-rosis but also by modifying the composition, structure, andstability of the cellular membrane. High levels of circulatingcholesterol and its accumulation in the heart tissue have beenassociated with cardiovascular damage [27]. Rats treatedwithISO showed a significant increase in the serum levels of TC,TGs, and LDL-C, as previously reported [28]. Generally, themechanisms of actions of lipolytic hormones including ISOon fat cells are believed to be mediated by the cAMP cascade,in which lipolytic hormones activate adenylate cyclase andthereby increase cAMP formation. Then, cAMP promoteslipolytic activity by activating cAMP-dependent proteinkinase, which phosphorylates hormone-sensitive lipase [29]and results in the hydrolysis of stored triacylglycerol, therebycontributing to marked hyperlipidemia [30]. High levels ofLDL-C have been positively correlated with MI, while highlevels of HDL-C have a negative correlation. HDL-C inhibitsthe uptake of LDL-C by the arterial walls and facilitates thetransport 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 ofthe myocardial membrane. The mechanism by which SHincreases serum HDL-C and decreases TC, TGs, and LDL-C levels is under investigation. The flavonoids such as rutinhave been identified in SH shown to inhibit platelet aggre-gation, anti-inflammatory effects, antioxidant, and reductionof blood fat and cholesterol level. On the other hand, nar-ingin has also been shown to exhibit cardioprotective effectthat might be via its cholesterol lowering and antihyper-glycemic activities [32]. Alagwu et al. [33] postulated thathoney increases bile cholesterol excretion and lowers plasmacholesterol levels. It is also plausible that honey reducesplasma cholesterol through its antioxidant properties and byenhancing the synthesis of HDL-C in the liver [34]. Polyphe-nols, particularly gallic acid and catechin, have been reportedto inhibit cholesterol esterase [35]. In general, pancreaticcholesterol esterase plays an important role in hydrolyzingdietary cholesterol esters, which liberates free cholesterol inthe lumen of the small intestine [36].Therefore, the inhibitionof cholesterol esterase is expected to limit the absorbance ofdietary cholesterol, resulting in reduced cholesterol absorp-tion. Moreover, polyphenols can also bind with bile acids toincrease their fecal excretion, which has been hypothesized as

a possible mechanism for the lowering of plasma cholesterollevels by SH [35].

In recent years, research has increasingly focused on freeradicals that may modify biological molecules and result invarious pathological conditions [37]. Lipid peroxidation, atype of oxidative deterioration of PUFAs, has been linkedwith altered membrane structure and inactivation. In ourstudy, ISO treatment resulted in a significant increase inthe levels of lipid peroxidation products in the heart tissue.Increased lipid peroxidation appears to be the initial stagein making the tissue more susceptible to oxidative damage.This may be responsible for the observed membrane damageevidenced by elevated lipid peroxidation levels [24].However,pretreatment with SH showed significantly lowered levelsof lipid peroxides in ISO-induced rats. The antioxidantnature of SH may hinder ROS production stimulated by ISOadministration and confer the protection to cardiac tissue.

The generation of ROS occurs due to the leakage of elec-trons from various systems into oxygen. Endogenous antiox-idant enzymatic defense is a very important step in the neu-tralization of oxygen free radical-mediated tissue injury [38].SOD, catalase, and GPx are the primary free radical scav-enging enzymes involved in the first line of cellular defenseagainst oxidative injury, removing O

2and H

2O2before they

can interact to formmore reactive hydroxyl radicals [39, 40].In this study, significantly lower SOD and GPx activities wereobserved in the heart tissues of ISO-induced rats comparedwith control rats. The observed decrease in the activities ofthese enzymes may be due to increased generation of ROSincluding hydrogen peroxide, superoxide, and hydroxyl rad-icals. This in turn leads to inactivation of these enzymes andresults in decreased removal of superoxide radicals, H

2O2,

and highly potent hydroxyl radicals [41, 42]. However, pre-treatment with SH improved the scavenging activities of SODand GPx for superoxide and H

2O2produced by ISO. GRx

is an antioxidant enzyme involved in the reduction of glu-tathione disulfide (GSSG) (an end product of the GPx reac-tion) to glutathione (GSH) [17]. In ISO-treated rats, a markedreduction in GPx activity was observed, leading to reducedavailability of the substrate for GRx, thereby decreasing theactivity of GRx. However, oral pretreatment of ISO-treatedrats with SH restored the activity of GRx, thereby acceleratingthe conversion of GSSG to GSH. The upregulation of theactivity or expression of Nrf2 is plausible; a transcriptionfactor released from its repressor (Keap1) under oxidative orxenobiotic stress [43] is considered as possible mechanismthrough which SH pretreatment restores antioxidant enzymefunctions as also suggested by Erejuwa et al. [44]. Thereleased Nrf2 binds to the antioxidant response element ofcytoprotective genes and induces their expression as well asthe subsequent expression of free radical scavenging enzymesto neutralize and eliminate the cytotoxic oxidants [43].

The improvements in biochemical parameters reportedin the current study were more pronounced than the histo-pathological results. Nevertheless, histopathological exami-nation of myocardial tissues in normal control rats illustratedclear integrity of the myocardial cell membranes and noinflammatory cell infiltration. Heart tissues from rats treated


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

Parameters Tualang honey treatment Percentage change (%)Sham TH + ISO ISO TH SH

cTn I (ng/mL) 0.06 ± 0.01 0.18 ± 0.05 0.69 ± 0.05 80.38 91.02CK-MB (U/L) 94.44 ± 3.05 158.03 ± 7.59 242.39 ± 7.30 57.02 60.89LPO (nmol TBARS/mg of protein) 9.25 ± 0.68 16.84 ± 1.06 22.42 ± 1.09 42.36 91.46SOD (units/mg of protein) 1.46 ± 0.05 0.19 ± 0.02 0.16 ± 0.01 2.31 53.25GPx (nmol NADPH oxidized/min/mg of protein) 2.86 ± 0.06 1.86 ± 0.20 1.07 ± 0.19 44.13 59.72TC 45.55 ± 2.49 56.77 ± 3.70 76.52 ± 4.84 63.77 75.82TGs 46.31 ± 3.68 53.37 ± 3.91 72.88 ± 3.28 73.41 99.75Results are expressed as the mean ± SD, 𝑛 = 10. Percentage of protection provided by each honey type is calculated as 100 × [(value of ISO group − value ofhoney + ISO group)/(value of ISO group − value of sham group)]. TH: Tualang honey and SH: Sundarban honey.

with ISO alone showed widespread myocardial structure dis-order, coagulative necrosis, cardiac muscle fiber separation,and inflammatory cell infiltration. However, the histopatho-logical findings of the SH-pretreated myocardial infractedhearts showed nearly normal morphologic cardiac musclecharacteristics and the absence of necrosis compared withhearts from rats treated with ISO alone. The reduced inflam-matory cell infiltration and cardiac muscle fiber architecturaldamage further confirmed the cardioprotective effects of SH.

Thefindings on cardioprotective efficacy of SHwere com-pared with Malaysian Tualang honey (TH) [41] for thepercentage changes in similar biological parameters (Table 5).SH showed better effects compared to TH when MI wasinduced by ISO at 85mg/kg body weight.

Overall, the probable mechanism by which SH exhibitedits protective and ameliorative effects against ISO-inducedmyocardial damage is via improved antioxidative status andlowered plasma cholesterol level. Further studies should beconducted to clarify the exact mechanism of the cardiopro-tective effect of SH.

5. Conclusion

Pretreatment with SH significantly altered nearly all bio-chemical parameters associated with ISO-induced myocar-dial injury. These findings were confirmed by histopatho-logical examination of normal and infracted rat hearts. Thisfindingmay provide scientific support to understand the ben-eficial effects of SH on cardioprotection against myocardialinjury, in which oxidative stress has long been known to con-tribute to the pathogenesis.

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

This research is partly supported by the Bangladesh MedicalResearch Council (BMRC) Grant 2012-2013 and partly byTWAS 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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