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European Journal of Applied Sciences – Vol. 13, No. 1
Publication Date: February 25, 2025
DOI:10.14738/aivp.131.15977.
Nkanu, E. E. (2025). Co-Administration of Naringenin with Isoproterenol Alters Cardio-Hepatic Bioactivity in Experimental Animal
Models. European Journal of Applied Sciences, Vol - 13(1). 419-426.
Services for Science and Education – United Kingdom
Co-Administration of Naringenin with Isoproterenol Alters
Cardio-Hepatic Bioactivity in Experimental Animal Models
Etah E. Nkanu
Kampala International University, Western Campus Uganda
Cross River University of Technology, Calabar, Department of Physiology
ABSTRACT
Isoproterenol is a beta-1 adrenergic agonist that has over time been used in the
management of cardiovascular cases. Above optimal dosage, it may lead to altered
liver function and cardiac activity shown by variations in heart and liver
biomarkers. This study aimed at investigating the effect of co-administration of
isoproterenol and naringenin on cardio-hepato activity and when administered at
separate treatment periods. Female Wistar rats were randomly divided into four
groups of five rats. Group A (Control) received normal saline, group B received
18mg/kg body weight of Isoproterenol in saline (given subcutaneously, for one
week), Group C received 18mg/kg of isoproterenol and naringenin (40mg/kg
orally), Group D received Isoproterenol (18mg/kg) for one week and supplemented
with naringenin for two weeks. Treatment lasted for 21days. Blood samples were
collected for biochemical analyses, heart and liver were collected for histological
analyses. Plasma Creatine kinase, Troponin, Lactate dehydrogenase,
Malondealdehyde, liver enzymes, all increased in isoproterenol administration
alone; dropped with co-treatment with naringenin except for ISO+NRG, but
increased in activity at 1week isoproterenol treatment and then followed by two
weeks naringenin treatment. Histological alterations were prominent in heart and
liver of rats in all treatment groups showing moderate aggregate of myocardial
inflammation with loss of cardiac tissues and also infiltration of inflammatory
hepatic cells with periductal fibrosis. This results suggest that co-administration of
isoproterenol with naringenin has in vivo efficacy and has the potential to improve
cardiac-hepatic function, but may fail to ameliorate tissue injury at separate
treatment periods.
Keywords: Isoproterenol, Troponin, Naringenin, Creatine kinase, liver enzymes, heart.
INTRODUCTION
Isoproterenol ([1-(3-4-dihydroxyphenyl)-2-isopropylaminoethanol hydrochloride)] is a
synthetic catecholamine and a non-selective β-1 adrenergic agonist that is used in the
management of cardiovascular disease. However, adverse effect has also been reported
especially in cases of overdose and acute toxicity (1). The adverse effect range from severe
stress in the myocardium resulting in compromised cardiac integrity and expression of infarct
like-necrosis [2] to subsequent generation of cytotoxic free radicals via autoxidation of
cathecholamines [3]. In event of overdose, contraindications or side effect occur such as
Hepatotoxicity and glycogenolysis, activation of renin angiotensin –aldosterone system in the
Kidney[4,5] intracellular depletion of ATP , elevated levels of calcium which activates beta-1
adrenergic receptor, increased myocardial cAMP and altered membrane permeability[6 ,7]
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European Journal of Applied Sciences (EJAS) Vol. 13, Issue 1, February-2025
with a resultant increase in cardiac-specific troponins, troponin I (cTnI) and troponin T (cTnT)
considered as biochemical markers in myocardial infarction, and unstable angina[8,9]
Generally, experimental findings have demonstrated that the actions of Isoproterenol (ISO) via
stimulation of the adrenergic receptors brings to bare the main index of the pathogenesis of
myocardial hypertrophy. This has been partly attributed to enhanced protein synthesis and
cardiac mass and exposes such vulnerable heart to ischemic injury [1]. However, in order to
ameliorate toxicity often caused by drugs and their negative physiological impact in the body,
the use of plant/herbal product is gradually being considered as an alternative medicine. The
high-profile phytochemical components in such plants and their antioxidant and anti- inflammatory activity [10,11] mediates their beneficial responses and essential for their
protective function. Naringenin, a naturally-occurring flavonoid of the flavanone subclass is
predominantly found in citrus fruits and tomatoes [12,13] and because of its dietary
antioxidant property, it has received a special attention. Naringenin exhibits various
pharmacological and therapeutic properties including anti-mutagenic, anti-inflammatory and
free radical scavenging activity [14,15]. The aim of this research work therefore, was to find out
the bioactivity of naringenin in isoproterenol-induced cardio-hepatic toxicity in experimental
animal models.
EXPERIMENTAL DESIGN & METHODS
Animals
Animals were obtained from the Department of Physiology, Faculty of Basic Medical Sciences,
Cross River University of Technology, Calabar, Nigeria. The animals were kept in standard cages
and under standard conditions with 12-h light/dark cycle, at 36°C ± 4°C. All the rats had free
access to food and water ad libitum. Ethical approval for the study and use of Laboratory
Animals was obtained from the Faculty of Basic Medical Science Animal Research Ethical
Committee of Cross River University of Technology, Calabar, Nigeria. Approval number
(CRUTECH/FBMS/NIG/REC./2023/25).
Experimental Design
Acclimatization period for the female Wistar rats was one week after which the animals
(weighing between 180-200g) were randomly selected and put into four cages containing five
rats each. Group A was Control. Group B received 18mg/kg body weight of Isoproterenol
(subcutaneously) only (ISO). Group C received 18mg/kg of Isoproterenol + 40mg/kg of
naringenin orally (ISO + NRG) (co-administration). Group D received 18mg/kg of ISO for 1week
and then NRG 40mg/kg body weight for two weeks (ISO 1wk + NRG 2wks). Isoproterenol
hydrochloride at 18mg/kg dissolved in normal saline (0.9 NaCl) was administered
subcutaneously for one week. The animals in group D were treated with isoproterenol for one
week after which NRG (40 mg/kg orally) was supplemented. After the last treatment of ISO and
NRG, animals were anesthetized and blood was immediately collected by cardiac puncture into
blood sample bottles and centrifuged at 1000 rpm for 10 minutes. The serum obtained was
refrigerated at 4°C and used for various biochemical evaluations.
Biochemical Analysis
Blood samples collected by cardiac puncture were put in plain bottles while the organs collected
were stored in tissue sample bottles and stored at 10o C and later used for biochemical analyses.
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Nkanu, E. E. (2025). Co-Administration of Naringenin with Isoproterenol Alters Cardio-Hepatic Bioactivity in Experimental Animal Models. European
Journal of Applied Sciences, Vol - 13(1). 419-426.
URL: http://dx.doi.org/10.14738/aivp.131.15977
Troponin, CK-MB and lactate dehydrogenase as well as liver enzymes such as Aspartate
aminotransferase (AST), alanine aminotransferase (ALT), and alanine phosphatase (ALP), were
determined using commercially available kits.
Serum Cardiac Marker Enzymes
The activity of serum cardiac marker enzymes like cardiac troponin C (cTnC), lactate
dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were assessed to check the cardiac
function and integrity. Serum levels of myocardial-damage enzyme markers were measured
from blood samples collected by cardiac puncture from the rat at the time of euthanasia.
Creatine phosphokinase (CK) (EC2.7.3.2) and its heart isoenzyme (CK-MB), as well as Troponin
were determined using conventional diagnostic kits. Aspartate aminotransferase (AST, EC
2.6.1.1), other liver enzymes and lactic dehydrogenase (LDH, EC1,1,1,27) were measured by the
method of (16,17).
Malondialdehyde:
Plasma MDA was estimated by method of (18). After the reaction of thiobarbituric acid with
malondialdehyde, the reaction product was extracted in butanol and was measured.
Histological Studies Tissue Processing for Histology
Following fixation, paraffin embedding and 4μm tissue sections (heart and liver) were obtained
with the Reichert Jung 2050 rotary microtome, followed by Haematoxylin and Eosin staining
procedures. A light microscope was used to examine the slides, and photomicrographs taken
with a digital camera (19). The slides were viewed at magnification of X 400 and
photomicrographs were taken.
STATISTICAL ANALYSIS
Statistical analysis was performed using Graph pad version 5.0. Results obtained are expressed
as mean±SEM. Comparisons between multiple groups were assessed using one-way analysis of
variance (ANOVA) and a post hoc test using Bonferroni multiple range test to detect significant
differences between groups. Statistical significance was accepted at the level of P<0.05.
DISCUSSION
The use of isoproterenol ([1-(3-4-dihydroxyphenyl)-2-isopropylaminoethanol hydrochloride)]
in the treatment of myocardial infarction and other related cardiovascular issues has being on
for years. It is a synthetic catecholamine, a βeta-1 adrenoceptor agonist found predominantly
in the heart. Apart from its role in the treatment of cardiovascular disease, such as congestive
heart failure and cardiogenic shock [1], our interest was to examine the possible ameliorative
effect of naringenin on the possible damage often caused by the drug (isoproterenol) in the
heart and liver in adverse dosages as well as alterations in cardiac enzyme activity and hepatic
biomarkers that occur in cardiovascular damage.
In this study, we have shown that high dose administration of isoproterenol induced cell
oxidative stress. Usually, persistent tissue stress triggered by disoriented mitochondrial
membrane and catecholamine auto-oxidation [20,21,22] results in a geometric generation of
free radicals or reactive oxygen species which induce tissue injury and may lead to the initiation
of pathologic alterations in the myocardium, including inflammation and cell death. This