Accepted Manuscript

Roselle is cardioprotective in diet-induced obesity rat model with

myocardial infarction

Lislivia Yiang-Nee Si, Siti Aishah Mohd Ali, Jalifah Latip,

Norsyahida Mohd Fauzi, Siti Balkis Budin, Satirah Zainalabidin

PII: S0024-3205(17)30544-1
DOI: doi:10.1016/j.lfs.2017.10.030
Reference: LFS 15399
To appear in: Life Sciences
Received date: 19 July 2017
Revised date: 11 October 2017
Accepted date: 20 October 2017

Please cite this article as: Lislivia Yiang-Nee Si, Siti Aishah Mohd Ali, Jalifah Latip,
Norsyahida Mohd Fauzi, Siti Balkis Budin, Satirah Zainalabidin , Roselle is
cardioprotective in diet-induced obesity rat model with myocardial infarction. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Lfs(2017), doi:10.1016/j.lfs.2017.10.030

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 ACCEPTED MANUSCRIPT

Roselle is cardioprotective in diet-induced obesity rat model with myocardial

infarction

Lislivia Yiang-Nee Sia, Siti Aishah Mohd Alib, Jalifah Latipb, Norsyahida Mohd Fauzic, Siti
Balkis Budina, Satirah Zainalabidina,*

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Affiliations:

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Program of Biomedical Science, School of Diagnostic and Applied Health Sciences, Faculty

of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300,
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Kuala Lumpur, Malaysia.
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School of Chemical Sciences and Food Technology, Faculty of Science and Technology,

Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia.

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Drug and Herbal Research Center, Faculty of Pharmacy, Universiti Kebangsaan Malaysia,
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Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia

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*Corresponding author
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Address : Program of Biomedical Science, School of Diagnostic and Applied

Health

Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia,

Jalan Raja Muda Abdul Aziz, 50300, Kuala Lumpur, Malaysia.

Email : satirah@ukm.edu.my

Tel No. : +603-92897684

Fax No. : +603-26929032

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ABSTRACT

Aims: Obesity increase the risks of hypertension and myocardial infarction (MI) mediated by

oxidative stress. This study was undertaken to investigate the actions of roselle aqueous extract

(R) on cardiotoxicity in obese (OB) rats and thereon OB rats subjected to MI. Main methods:

Male Sprague-Dawley rats were fed with either normal diet or high-fat diet for 8 weeks. Firstly,

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OB rats were divided into (1) OB and (2) OB+R (100 mg/kg, p.o, 28 days). Then, OB rats were

subjected to MI (ISO, 85 mg/kg, s.c, 2 days) and divided into three groups: (1) OB+MI, (2)

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OB+MI+R and (3) OB+MI+enalapril for another 4 weeks. Key findings: Roselle ameliorated

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OB and OB+MI’s cardiac systolic dysfunction and reduced cardiac hypertrophy and fibrosis.

The increased oxidative markers and decreased antioxidant enzymes in OB and OB+MI groups
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were all attenuated by roselle. Significance: These observations indicate the protective effect of

roselle on cardiac dysfunction in OB and OB+MI rats, which suggest its potential to be
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developed as a nutraceutical product for obese and obese patients with MI in the future.
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Keywords: Obesity, oxidative stress, myocardial infarction, roselle, cardiac function

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1. Introduction
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The incidence of cardiovascular diseases continues to grow across the globe,

contributing to the largest rate of mortality and morbidity each year [1]. Obesity is believed to

be one of the major pathophysiological contributor to initiation of cardiovascular diseases, such

as type II diabetes, dyslipidemia and hypertension [2], and it is closely related to the increased

risk of myocardial infarction (MI) [3]. In Malaysia, the rate of obesity has been found to be the

highest within Asian region, whereby 45.3 % of its population are overweight [4]. The World

Health Organization (WHO) has reported that at least 2.8 million of adults die each year as a
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result of being obese [5]. These data have clearly indicated that obesity has reached alarming

proportion in adults and even in children. Obesity is characterized with body mass index (BMI)

equal to or greater than 30 kg/m2 [6] and is significantly correlated with high plasma leptin

level [7]. It has been shown that long term of high fat diet (HFD) consumption in rats could

increase body weight and visceral fat/weight ratio, serum triglyceride, cholesterol and

myocardial hypertrophy, all of which consistent with human obesity [8-9].

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MI or commonly known as heart attack, is defined as necrosis of heart muscle,

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secondary to prolonged ischemia or hypoxia. It is one of the leading causes of death and

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disability in developed and developing countries, including Malaysia [10]. In the year 2010,

24.5 % of death in Malaysian government hospitals was due to cardiovascular diseases (CVD),
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where MI was ranked first among the leading cause of CVD mortality [11]. MI could lead to

progressive heart failure when pathophysiological adverse cardiac remodelling is developed.

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Distortion of left ventricular (LV) shape, LV dilatation, cardiomyocytes hypertrophy, scar

formation and increased wall stress could lead to LV systolic and diastolic dysfunction [12-13].
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Roselle or scientifically known as Hibiscus sabdariffa Linn., is a local tropical plant

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which is usually consumed as hot tea or juice due to its wide range of nutraceutical benefits.
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Roselle calyces are a rich source of high antioxidant polyphenols which contributes to its well-
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established antihypertensive properties [14]. A recent study reported protective effects of

roselle polyphenols against hyperglycemia and hyperlipidemia in insulin-resistant rats [15]. In

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addition, roselle aqueous extract supplementation showed protective effects against red blood

cell and cardiac oxidative damage in streptozotocin-induced diabetic rats and nicotine-induced

cardiac injury rats, respectively [16-17]. Moreover, roselle polyphenols were shown to

improve cardiac dysfunction and vasodilation, possibly via modulation of intracellular calcium

entry, release and reuptake in the heart [18]. While many studies have reported the

cardioprotective role of roselle, effects of roselle on obese and post-MI in obese rats has not
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been shown. Therefore, this study was designed to investigate the actions of roselle

supplementation on cardiac function, oxidative stress and remodelling in obese and post-MI of

obese rat models.

2. Materials and methods

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2.1 Roselle aqueous extract preparation

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Dried roselle (H. sabdariffa Linn. UMKL-1) calyces were purchased from HERBagus

Sdn Bhd in Kepala Batas, Malaysia with voucher specimen of PID050515-05 from Forest
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Institute of Malaysia (FRIM). Roselle aqueous extract was prepared based on method by
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Nnamonu et al. [19] with water ratio suggested by Chrumsri et al. [20]. The dried calyces were

ground into powder with blender (Cornell, Malaysia) and soaked in distilled water in 1:10 ratio

for 24 hours with occasional shaking. The mixture was then filtered with a piece of clean,
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sterile coarse cloth. Then, the filtrate was kept frozen at -20°C in an aluminium-wrapped
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plastic container overnight. The solidified roselle extract was then freeze-dried (Labconco, US)

and stored in a dark bottle at 4°C until use.

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2.2 Identification and quantification of anthocyanins

Anthocyanins were analysed by the high performance liquid chromatography (HPLC)

method. 10 mg of freeze-dried extract from roselle calyces were dissolved in 0.1 % formic acid

in water. The mixture was sonicated for 5 minutes and filtered using a 0.45 μm PTFE

membrane syringe filter (Gema Medical, Spain) prior to injection. HPLC was carried out using
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a HPLC Waters e2695 separation module equipped with a degasser (Waters, UK), an

autosampler automatic injector and a Waters 2998 Photodiode Array Detector. The separation

was performed using Purospher STAR RP-18e LichroCART column (250mm x 4.6mm x 5um

particle size) (Merck Chemicals, Germany) at a flow rate of 1.0 mL/min, injection volume of

20 μL and 30 °C column oven temperature. 0.1 % formic acid in water and 0.1 % formic acid

in acetonitrile were employed as mobile phases A and B respectively, in gradient elution as

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follows: 10-25 % B (0-5 min), 25-90 % B (15-20 min), 90-10 % B (25-25 min) and 10 % B

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(25-30 min). UV absorption was measured at 520 nm. Two compounds including delphinidin-

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3-O-sambubioside and cyanidin-3-O-sambubioside were used as standards.
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2.3 Chemicals and Drugs
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Anthocyanin standards (delphinidin-3-O-sambubioside and cyanidin-3-O-sambubioside)

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were purchased from Extrasynthase, France. All chemicals were of analytical grade. HPLC
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grade of solvents such as methanol and acetonitrile were obtained from Merck Chemicals,
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Germany while formic acid was obtained from Merck Chemicals, Finland. Isoproterenol
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hydrochloride and heparin were obtained from Sigma Aldrich, USA while urethane and

enalapril were obtained from Merck Chemicals, USA.

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2.4 Animals

All the procedures involving use of animals were subjected to approval from Universiti

Kebangsaan Malaysia Animal Ethics Committee. Healthy male Sprague-Dawley rats (7-8
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weeks; 250-300 g) were allowed to adapt to laboratory conditions for 1 week with ad libitum

access to water and normal rat diet before experiment started. Throughout the experiment, all

rats were housed under same laboratory conditions of ambient room temperature and lighting

(12 hours light dark cycle).

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2.5 Study design

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The rats were randomly allotted into 2 dietary groups to receive either normal diet (n=6)

(control, a diet containing 3% of fat, 22% of protein, 5% of fiber, 13% of moisture, 8% ash and
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49% of nitrogen free extract; Gold Coin, Malaysia) or high fat diet (HFD) (n=12) (a diet

containing 60% of fat, 20% of protein and 20% of carbohydrate; Research Diets, US). Duration
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for establishment of obese (OB) rats was 8 weeks [21] and validated by increased of plasma

leptin level compared to control. After the 8th week, all OB rats were subdivided into two
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subgroups (n=6 per group) to receive either vehicle (OB group; 0.9 % normal saline in an equal
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volume) or roselle aqueous extract (OB+R group; 100 mg/kg roselle aqueous extract) for 28

days [14] via oral-force feeding with continued diet. Throughout the experiment, body weight
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gained and food intake were recorded at weekly interval. In addition, heart weight/tibia length

(HW/TL) ratio were recorded at the end of experiment.

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2.6 Induction of MI in obese rats model

Another subset of study was carried out to investigate the actions of roselle in post-MI

of OB rats heart. After being fed with HFD for 8 weeks, these OB rats were given
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isoproterenol injection (ISO, 85 mg/kg, subcutaneous, 2 days) [22] to induce MI. OB rats with

successful induction of MI (OB+MI), which were validated by increased plasma Troponin-T

level, were subdivided into 3 subgroups (n=6 per group) to receive the following treatments for

28 days via oral forced feeding: (1) vehicle (OB+MI group; 0.9 % normal saline in an equal

volume), (2) roselle aqueous extract (OB+MI+R group; 100 mg/kg) [14] and (3) angiotensin

converting enzyme (ACE) inhibitor enalapril (OB+MI+E; 10 mg/kg) [23] which served as a

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positive control.

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2.7 Systemic characteristics determination in OB rats model
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Blood samples were collected by orbital sinus at baseline, week 8 and at the end of
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experiment (week-12) for plasma leptin monitoring using commercial ELISA kit (Cusabio

Biotech Co Ltd, China). Plasma cholesterol, triglycerides and high-density lipoprotein (HDL)
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level were determined at the end of experiment using a colorimetric assay kit (Biosystem
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Diagnostics, Spain). Plasma low-density lipoprotein (LDL) level was calculated using

Friedewald equation [24].

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2.8 Blood pressure measurement in OB rats and OB+MI rats models

Blood pressure (BP) was measured in conscious restrained rats at the end of the

experiment by using non-invasive tail-cuff method (CODA 2-channel non-invasive blood

pressure system, Kent Scientific Corporation, USA). Rats were placed in a container holder to

restrict its movement and placed on a warm plate for at least 5 minutes to increase blood flow
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in rats before BP measurement was performed. An occlusion cuff (O-cuff) was attached to rat’s

tail to constrict the tail artery while a volume pressure-recording (VPR) cuff detected changes

in tail artery volume when blood flow resumes as the O-cuff deflates. Each measurement

session consisted of 5 acclimatization cycles followed by 15 BP measurement cycles. A set

was accepted and blood pressure value including systolic BP and heart rate were recorded if the

system identified >50% successful readings.

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2.9 Isolated Langendorff-perfused rat heart

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Rats were injected with anticoagulant heparin (500 IU, i.p.) to prevent blood clot in the

heart followed by anesthesia with urethane injection (1 g/kg, i.p.). After the rat became
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unconscious and lost pedal reflex activity, the heart was excised. The isolated heart was then

placed in ice-cold Krebs-Henseleit buffer and cannulated via aorta to Langendorff apparatus
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immediately. The heart was perfused retrogradely at constant pressure mode (80 mmHg) with
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Krebs-Henseleit buffer (in mM: NaCl 118.0; KCl 4.7; MgSO4 1.2; NaHCO3 25.0; KH2PO4 1.2;

CaCl2 2.5; glucose 11.0) of pH 7.4, which was continuously aerated with 95 % O2 and 5 %
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CO2 at 37 °C. A water-filled latex balloon connected to a pressure transducer (MLT844,

ADInstrument, Australia) was then inserted through mitral valve into left ventricle to allow
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isovolumetric contraction in order to measure intraventricular pressure changes. After 20

minutes of stabilization, cardiac function parameter such as left ventricular developed pressure

(LVDP), left ventricular (LV) maximum and minimum rate of pressure changes (LVdP/dt max

and LVdP/dtmin) as well as the time constant of isovolumic relaxation (Tau) were assessed

using a PowerLab data acquisition system and analysed using chart software (LabChart 7.0,

ADInstrument, Australia). The rate of coronary flow was also recorded by measuring the
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amount of perfusate flow out from coronary in one minute., whereas rate of pressure product

(RPP) was formulated from LVDP x HR.

2.10 Heart homogenate preparation and oxidative stress assessment

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A portion of heart tissue was homogenized in phospholysis buffer solution in a ratio of

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1:3 (w/v). After centrifugation at 12,000 rpm for 30 minutes at 4 °C, supernatant was collected

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and kept frozen at -80 °C for oxidative stress assessment by measuring 8-isoprostane level

using commercial ELISA kit (Elabscience Biotech Co Ltd, China). In addition, endogenous
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antioxidant status in heart homogenate was also determined by measuring superoxide

dismutase (SOD) enzyme activity [25] and reduced glutathione (GSH) concentration [26] using
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colorimetric assay methods.

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2.11 Cardiac histological analysis

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A small portion of heart tissue was fixed in 10 % formalin and embedded into paraffin
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blocks. Paraffin-embedded tissues were sectioned into 4 µm thickness and stained with

Hematoxylin and Eosin (H&E) in order to measure size of cardiomyocytes and Picrosirius Red

in order to estimate percentage of collagen deposition (fibrosis). Quantitative measurement of

the heart, including cardiomyocyte cross-sectional area was calculated by measuring

circumferential length of myocytes while percentage of collagen deposition was calculated

using Sirius Red Macro software. Analysis for both measurements were done using ImageJ

software (Bethesda, Maryland, USA).

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2.12 Gene expression analysis

RNA was extracted from frozen heart tissue and reverse transcribed using previously

described methodology [27]. Gene expression of oxidative stress marker (NADPH oxidase

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subunit, Nox2), hypertrophy marker (atrial natriuretic peptide, ANP) and fibrosis marker (brain

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natriuretic peptide, BNP) were measured via quantitative real time-PCR using SYBR® Green

(Applied Biosystems, Scoresby, Victoria, Australia). In addition, ribosomal 18S gene

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expression was used as the endogenous control. Primers were generated from rat sequences in
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GenBank. Primer sequences are given in online-only supplement (Supplementary Table 1).

Quantitative analysis was performed using ABI Prism® 7700 Sequence Detection software,
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using the ΔΔCt method to detect fold differences relative to control group.
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2.13 Statistical analysis

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Values from each group were presented as mean ± SEM. Statistical significance was

determined by a one-way analysis of variance (ANOVA) followed by a Tukey’s post-hoc test

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to analyse differences between groups using GraphPad Prism software version 6. A probability

value (p-value) of less than 0.05 was considered as statistically significant.

3. Results
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3.1 Anthocyanins were characterised from roselle aqueous extract

Two anthocyanin compounds were detected from roselle extract by HPLC.

Identification of each single compound was mainly based on HPLC retention time and

matching UV absorption spectrum with standard compounds at 520 nm. Anthocyanins

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quantification was determined by standard reference calibration curves and was expressed as

mg per g freeze dried extract (FDE). Linear correlation co-efficient were > 0.996 for each

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compound. The result showed that roselle calyces contained delphinidin-3-O-sambubioside

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(6.40 min) and cyanidin-3-O-sambubioside (7.98 min) with 7.10 ± 0.02 and 2.91 ± 0.03 mg /g

FDE respectively (Fig.1).

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3.2 Systemic characteristics in OB and OB+MI rat models

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After 12 weeks of HFD consumption, data showed that body weight gain, HW/TL ratio,
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plasma leptin, cholesterol, triacylglycerol, LDL and systolic blood pressure were significantly
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(p < 0.05) increased in OB and OB+MI groups rats compared to control group (Table 1).

Roselle and enalapril treatments were able to significantly (p < 0.05) attenuate all these
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systemic characteristics except for HW/TL value (p = 0.7423 in OB+R group compare to OB

group, p = 0.7310 in OB+MI+R group while p = 0.0623 in OB+MI+E group compared to

OB+MI group, Table 1). Food intake and heart rate remain unaltered by OB or roselle

treatment (Table 1). However, heart rate in OB+MI group was significantly (p < 0.05)

increased compared to control group and was significantly (p < 0.05) attenuated by roselle and

enalapriltreatments (Table 1).

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3.3 Roselle supplementation ameliorates cardiac dysfunction in Langendorff-perfused

OBand OB+MI rat models

OB and OB+MI groups showed significant (p < 0.05) deterioration in cardiac systolic

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function (LVDP and LVdP/dtmax), diastolic function (LVdP/dtmin and Tau),coronary flow as

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well as cardiac output (RPP) compared to control group (Fig. 2). However, roselle and

enalapril treatments effectively ameliorated cardiac systolic dysfunction and rescued coronary

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flow and cardiac output (Fig. 2A, B, E and F). Besides that, treatment with roselle also showed
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a tendency to ameliorate cardiac diastolic dysfunction, although this was not statistically

significant (p = 0.3953 in OB+R group compared to OB group, p = 0.0579 in OB+MI+R group

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compared to OB+MI group, Fig. 2C and p = 0.1402 in OB+R group compared to OB group,

Fig. 2D), different from treatment with enalapril which showed statistically significant (p <
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0.05) (Fig. 2C and D).

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3.4 Roselle supplementation reduces myocyte hypertrophy in OB and OB+MI rat models
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Cardiomyocytes cross-sectional area which was measured using H&E-stained showed

significant (p < 0.05) enlargement of OB and OB+MI compared to control rats’ hearts (Fig.

3B). Gene expression of myocyte hypertrophy marker (ANP) was increased in OB group

compared to control group, although it was not statistically significant (p = 0.1765, Fig. 3C).

The gene expression was also increased in OB+MI group and it was statistically significant (p

< 0.05) compared to control group (Fig. 3C). Treatments with roselle and enalapril markedly
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reduced the cardiomyocytes cross-sectional area (Fig. 3B). Representative images for H&E

stained section is shown in Fig. 3A.

3.5 Roselle supplementation reduces cardiac fibrosis in OB and OB+MI rat models

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OB and OB+MI hearts exhibited increased interstitial collagen deposition as measured

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using Picrosirius-Red staining compared to control rat hearts (p < 0.05) (Fig. 4B). However,

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the cardiac fibrosis marker (BNP) gene expression was not significantly increased (p = 0.1118

in OB group, p = 0.0541 in OB+MI group, Fig. 4C). Treatments with roselle and enalapril
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markedly reduced the cardiac fibrosis (as shown in Fig. 4B). Representative images for

Picrosirius-Red stained section is shown in Fig. 4A.

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3.6 Roselle supplementation attenuates oxidative stress in OB and OB+MI rat hearts
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Obesity causes oxidative stress, as shown by increased Nox2 gene expression and 8-
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isoprostane level, as well as decreased endogenous antioxidants SOD activity and GSH
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concentration in the OB rats heart compared to control rats heart. These conditions were

similarly seen in the OB+MI hearts. All of these conditions could be attenuated by treatments

with roselle and enalapril (p < 0.05 for all, Fig. 5A-D).

4. Discussion
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In the present study, we have demonstrated for the first time that treatments with roselle

and enalapril for 28 days successfully (i) ameliorated cardiac function, (ii) reduced

cardiomyocyte hypertrophy and cardiac fibrosis, and (iii) attenuated cardiac oxidative stress in

obesity alone as well as in both obesity with MI conditions.

Using an established rodent model of obesity that resulted from administration of HFD

consumption, we were able to show that obesity is associated with systemic characteristics of

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high body weight gain, plasma leptin, lipid profile as well as high blood pressure. Interestingly,

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the present study showed that roselle successfully attenuate all these abnormalities. These

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results are consistent with previous studies which reported that roselle was effective in

alleviating weight gain [28], hyperlipidemia [15] and hypertension [14]. However, roselle did
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not alter the plasma HDL significantly in spite of the reducing effect in LDL. This is

presumably due to roselle’s capability in LDL’s hydrolysis, but not on the reverse cholesterol
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transport of HDL [29]. Leptin is an obesity hormone that is primarily secreted by adipose tissue

[30]. Its level has been reported significantly correlated to total body fat [7] and thus it has
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been used as a biomarker for validation of obesity in the present study. The mechanism by
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which roselle reduced plasma leptin level is not fully elucidated. However, a previous study
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suggested that polyphenols contained in roselle, particularly hydroxycitric acid and hibiscus
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acid were able to increase excretion of lipid content in feces, thus reducing lipid and adipose

tissue accumulation in the body [31]. Another study also showed that roselle inhibited lipid
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accumulation in adipocytes by reducing gene expression of adipogenic transcriptional factors,

such as PI3K Akt and ERK which play an important role in adipogenesis [32]. We have shown

in preliminary investigation that roselle on its own does not affect blood pressure, body weight,

plasma LDL as well as oxidative stress (Supplementary Table 2).

In addition to changes in systemic characteristics, we have shown that diet-induced

obesity markedly impaired the systolic function. We demonstrated for the first time that
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treatment with roselle effectively ameliorates systolic dysfunction of rat hearts, as clearly

evident by a significant rise in LVDP and LVdP/dtmax. Besides that, obesity was found to be

associated with significant fall in LVdP/dtmin, accompanied by rise in Tau, indicating that

ventricular chamber had stiffened [33]. However, the trend changes to both LVdP/dtmin and

Tau by roselle treatment suggested that progression of heart failure was lifted. Previous study

have reported that HFD administration could increase lipid profile in heart, indirectly

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indicating that there is lipid accumulation in coronary vessel which block coronary circulation,

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thus causing fall in coronary flow rate [34]. Besides rescued coronary flow, treatment with

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roselle was able to improve RPP significantly, a comparable indicator of cardiac output as it

reflects the heart’s workload or oxygen demand [35].

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Apart from impaired cardiac function, abnormal cardiac remodeling is also a key stage

of heart failure. It is characterised by cardiomyocyte hypertrophy and increased deposition of

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cardiac collagen [36-37]. In the present study, we demonstrated that obesity was able to induce

cardiac remodeling, as evidenced by a significant increase in cardiomyocyte cross-sectional

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area and increases trend of ANP gene expression. ANP is a type of natriuretic peptide, which is
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upregulated in heart during hypertrophy and heart failure as a result of hypertension [38]. In
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addition to that, obesity appears to cause cardiac fibrosis, similarly reported by previous studies
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[39-40]. Consistent with an earlier study, we have shown that treatment with roselle reduces

cardiomyocyte hypertrophy and tended to lower ANP gene expression, suggesting roselle may
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have anti-hypertrophic property [41]. This finding may be in part due to reactive oxygen

species (ROS) scavenging effect, whereby ROS is strongly linked with pathogenesis of cardiac

hypertrophy [42]. Besides alteration in the size of cardiomyocytes, it was of interest to note

that treatment with roselle reduced cardiac interstitial collagen accumulation, indicated by

reduction of BNP gene expression, a cardiac fibrosis biomarker [43]. Accumulation of collagen

in the heart, particularly collagen 1, could significantly affect ventricular compliance, reduce
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elasticity of ventricle wall and therefore contributes to impaired relaxation [44]. Therefore,

reduction of cardiac fibrosis and BNP gene expression by roselle could improve ventricular

compliance and relaxation. This finding suggests that the improvement in cardiac diastolic

function from roselle treatment was possibly due to reduced interstitial fibrosis.

Cardiac oxidative stress has been widely reported as a contributing factor for cardiac

dysfunction and remodeling [12]. A major source of reactive oxygen species (ROS) in cardiac

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tissue is NADPH oxidase system, where alteration in gene expression of NADPH oxidase

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subunits Nox-2 could contribute to oxidative damage in the heart [45]. Induction of obesity

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was found to significantly increase the gene expression of Nox-2 and production of 8-

isoprostane, markers of oxidative stress [46-47]. Nox-2 is a membrane-bound enzyme complex

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which generates superoxide anion when activated in response to obesity induction [48]. Upon

activation, Nox-2 has been shown to induce 8-isoprostane production via promoting lipid
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peroxidation process. Supplementation of roselle for 28 days was found to suppress gene

expression of Nox-2 and also production of 8-isoprostane.

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In parallel to oxidative stress mechanism, obesity also depletes antioxidant status in

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cardiovascular system as evidenced by reduction in SOD enzyme activity and GSH

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concentration. Free radical scavenger enzymes, such as SOD, are the first line of defense
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against oxidative injury, decomposing superoxide anion to water and oxygen. Similarly, GSH

is important in protecting myocardium against damage by free radicals [49]. In this study, we
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demonstrated a depletion in both antioxidant SOD and GSH status in obese rat hearts compared

to control, similar to previous study using HFD-induced obesity rats [47]. This suggests that

the cells have lack of capacity to neutralize the accumulation of ROS [50]. Cytotoxic ROS was

shown to upregulate expression of beta-transforming growth factor (TGF-β) in the heart and

promote proliferation of fibroblast and accumulation of collagen, which eventually contribute

to cardiac remodeling and cardiac dysfunction [45]. However, treatment with roselle was able
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to restore the antioxidant status, as similarly reported by previous study [51]. Antioxidant

property of roselle could be due to its anthocyanins content, such as delphinidin-3-O-

sambubioside and cyanidin-3-O-sambubioside, as seen in our roselle’s HPLC profiling. The

structure of these anthocyanins were reported to consist of an o-difenol structure in ring-B and

a conjugated double bond system, which serves to stabilize free radicals by donating electrons

to free radicals [52]. This would consequently help to protect the cells from free radicals attack

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and increase the integrity and function of the cell [16]. Other than that, as mentioned earlier,

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our results also suggested that attenuation of oxidative stress in the heart was dependent on

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improvement of systemic characteristics, such as hyperleptinemia and hyperlipidemia, which

are the key contributors to ROS production [53].

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This study was further investigated to determine protective actions of roselle in both

obese and obese with MI conditions. The present study has successfully indicated that obesity
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condition is worsened with MI in rats. In the ISO-administrated obese rat, cardiac systolic

dysfunction was clearly evident by a significant fall in LVDP and LVdP/dtmax, consistent to
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previous study [46]. Besides that, LVdP/dtmin was even markedly depressed, indicating a more
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severe diastolic dysfunction in obese rats following MI, which resulted in the persistence of
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elevated Tau [46]. In addition, cardiac remodeling features, such as myocyte hypertrophy and
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cardiac fibrosis were more severe in obese with concomitant MI than in obese alone. This was

further confirmed by the increase in gene expression of ANP and BNP. The mechanism by
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which cardiac dysfunction is more severe in OB with MI condition than obese without MI, is

due to a more prominent elevation of oxidative stress, as shown by increased in gene

expression of Nox-2 and production of 8-isoprostane, and a greater reduction in antioxidant

SOD and GSH status. Following that, treatment with roselle was capable to give protective

effect to obese rats heart given ISO, comparable to enalapril treatment, an ACE inhibitor,

which is clinically used to manage MI [54-55]. Favourable actions of enalapril in this study
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may have resulted from both decreased level of vasoconstrictor peptide angiontensin II and

also accumulation of bradykinin [56]. Enalapril treatment attenuated declining cardiac function

in obese and obese with MI rats, possibly via reduction of blood pressure which then unloads

the heart. Consistent with previous studies, enalapril also successfully ablated cardiac fibrosis,

cardiomyocyte hypertrophy [57] and oxidative stress within myocardium of rats in this study

[58]. In addition, circulating leptin were greatly reduced in enalapril treated rats [56], indicative

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of potent adipocyte loss that may benefit cardiovascular system. Interestingly, effect of roselle

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on cardiac fibrosis and hypertrophy were highly comparable to enalapril treated group,

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suggesting that roselle is as effective as ACE inhibitor which are commonly used to manage

MI complication.
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5. Conclusion
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This study demonstrates for the first time that roselle is effective in ameliorating
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cardiac function, reducing myocyte hypertrophy and cardiac fibrosis as well as attenuating
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oxidative stress. These findings suggest roselle has high potential to be developed into a
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nutraceutical product to reduce cardiovascular disease risk in obese patients. In addition, we

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have also shown that the protective effects of roselle is comparable to the ACE inhibitor,

enalapril. This suggests that roselle may also provide benefit as an adjunct supplement for
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obese patients affected by myocardial infarction. On a separate note, the cell signaling

mechanisms underlying protective actions of roselle against myocardial infarction in obese

condition require further investigations.

Conflicts of interest
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The authors declare no conflicts of interest.

Acknowledgements

This study was supported by NKEA Research Grant Scheme (NRGS) from Ministry of

Agriculture (MOA) (NH1014D061), Malaysia. We are thankful to Faculty of Pharmacy, UKM

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for lending their Langendorff apparatus, bachelor student Miss Rumaisak Farizal for her

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assistance in animal handling and postgraduate student Mr Anand Ramalingam for technical

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assistance in PCR.
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Table and Figures

Table 1

Systemic characteristics after 12 weeks HFD consumption. Values are presented as mean ±
SEM for n=6 per group. ap < 0.05 vs. Control, bp < 0.05 vs. OB, cp < 0.05 vs. OB+MI.

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Parameter Control OB OB+R OB+MI OB+MI+R OB+MI+E

Body weight gain 50.00 ± 120.17 ± 95.45 ± 108.90 63.68 ± 46.77 ±

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(g) 6.96 6.13a 9.98a,b ± 90a 9.12a,c 5.10a,c

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Food intake 107.88 ± 114.51 ± 101.87 ± 117.17 106.20 ± 99.58 ±
(g/week) 3.13 12.87
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HW/TL (g/cm) 0.25 ± 0.31 ± 0.27 ± 0.31 ± 0.28 ± 0.01 0.25 ± 0.01
0.02 0.02a 0.01 0.02a
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Plasma leptin 70.45 ± 261.30 ± 196.41 ± 226.15 180.26 ± 174.94 ±

(ng/ml) 9.56 24.59a 14.16a,b ± 11.23a 16.64a,c 4.73a,c
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Plasma 2.20 ± 3.02 ± 2.08 ± 2.93 ± 2.26 ± 2.14 ±

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cholesterol 0.09 0.30a 0.13b 0.07a 0.04c 0.04c

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(mmol/L)

Plasma 0.97 ± 1.44 ± 1.19 ± 1.43 ± 1.10 ± 1.06 ±

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triglycerides 0.09 0.06a 0.06b 0.04a 0.02c 0.04c

(mmol/L)
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Plasma HDL 1.48 ± 0.89 ± 1.16 ± 0.96 ± 1.05 ± 1.13 ± 0.12

(mmol/L) 0.10 0.05a 0.15a 0.07a 0.04a

Plasma LDL 0.28 ± 1.47 ± 0.60 ± 1.31 ± 0.71 ± 0.52 ±

(mmol/L) 0.04 0.30a 0.12b 0.09a 0.07a,c 0.14c

Systolic BP 129.47 ± 178.60 ± 140.50 ± 214.72 162.20 ± 152.76 ±

(mmHg) 2.59 1.46a
2.13 a,b ± 6.36a 5.32a,c 5.46a,c
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Heart rate (bpm) 304.17 ± 343.33 ± 321.11 ± 364.80 325.20 ± 320.42 ±

3.53 19.46 6.26 ± 12.28a 9.00c 12.70c

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Fig.1 HPLC chromatograms of two target components in standards recorded at 520 nm.
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Quantification showed that roselle calyces contained (1) 7.10 ± 0.02 mg/g FED dephinidin-3-
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O-sambubioside (6.40 min) and (2) 2.91 ±0.03 mg/g FDE cyanidin-3-O-sambubioside (7.98
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min).
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Fig. 2 Roselle supplementation ameliorates cardiac dysfunction in Langendorff-perfused

OB and OB+MI rat model. (A) LVDP, (B) LVdP/dtmax, (C) LVdP/dtmin, (D) Tau, (E)
coronary flow and (F) RPP. Data are presented as mean ± SEM for n=6 per group. ap < 0.05 vs.
Control, bp < 0.05 vs. OB, cp < 0.05 vs. OB+MI.
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Fig. 3 Roselle supplementation reduces cardiomyocyte hypertrophy in OB and OB+MI

rat models. Representative images from microscopic observation of heart tissue for each
group with (A) H&E staining; 40x magnification . Measurement were taken from each group
for (B) cardiomyocytes cross-sectional area from H&E staining. Fold changs in gene
expression of (C) ANP, relative to the housekeeper 18S. Data are presented as mean ± SEM
for n=6 per group. ap < 0.05 vs. Control, bp < 0.05 vs. OB, cp < 0.05 vs. OB+MI.
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Fig. 4 Roselle supplementation reduces cardiac fibrosis in OB and OB+MI rat models.
Representative images from microscopic observation of heart tissue for each group with (A)
picrosirius red staining; 10x magnification . Measurement were taken from each group for (B)
percentage of collagen deposition from picrosirius red staining. Fold change in gene
expression of (C) BNP, relative to the housekeeper 18S. Data are presented as mean ± SEM
for n=6 per group. ap < 0.05 vs. Control, bp < 0.05 vs. OB, cp < 0.05 vs. OB+MI.
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Fig.5 Roselle supplementation attenuates oxidative stress in OB and OB+MI rat hearts.
(A) Nox2 fold changes in gene expression, relative to housekeeper 18S, measurement of
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oxidative stress marker, (B) 8-isoprostane and level of endogenous antioxidants (C) SOD and
(D) GSH. Data are presented as mean ± SEM for n=6 per group. ap < 0.05 vs. Control, bp <
0.05 vs. OB, cp < 0.05 vs. OB+MI.
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