INTRODUCTION
Metabolic
diseases often involve abnormal lipid and glucose metabolism, increasing the
risk of cardiovascular disease (Yan et al., 2021). Cardiac energy metabolism
disorders significantly impact heart function and structure (Federico et al., 2021). Under normal conditions, the
myocardium uses fatty acids and glucose, but under pathological conditions,
fatty acid metabolism changes significantly, leading to mitochondrial damage
and serious heart damage. Patients with metabolic syndrome or diabetes have
been found to have increased lipid uptake and utilization, resulting in fat
accumulation in the heart (Costantino et al., 2019; Gao et al., 2020).
Lipid profile consists mainly of total
cholesterol, triglyceride, LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C),
which are classified as diagnostic tool for cardiovascular risk factors (Dasgupta & Wahed, 2021). Lipids are described as the
significant building blocks of life and are the major substances for mammalian
cell function (Dasgupta & Wahed, 2021). Cholesterol is essential in the
progression of cardiovascular disease and it increases the risk of developing
atherosclerotic cardiovascular disease (CVD) (Lee & Siddiqui, 2022). Dyslipidemia has been linked to
diabetes mellitus, which is a modifiable risk factor for cardiovascular
complications in type 2 DM (Haile & Timerga, 2020). However, it is characterized by hypertriglyceridemia, reduced HDL cholesterol
levels, and an increased concentration of Low-Density-Lipoprotein particles (Daya et al., 2017; Hirano, 2018).
Electrolytes play critical roles in the body
fluid regulation and a variety of other biological processes (Maeda et al., 2016; Shrimanker and Bhattarai 2021). However, the principal electrolytes
of significance are sodium, potassium, and chloride, which go along with
magnesium, calcium, phosphate, and bicarbonate (Shrimanker and Bhattarai 2021).
Thus, an imbalance in the aforementioned electrolytes above either a low or
high levels could disrupt normal physiological processes of life, leading to
complex situations that threaten life (Shrimanker and Bhattarai 2021).
Electrolyte disturbances play a role in a variety of illnesses as underlying
disorders, pathophysiological changes caused by diseases and traumas, and
changes or complications following insults (Maeda et al., 2016).
Calcium is a significant electrolyte, which
forms an integrative compartment of the human body and has significance for
human health. It is known for the formation and maintenance of bone (Li et al., 2018). Plasma calcium is known for several
homeostatic role in the maintenance of skeleton, hormone regulation, nerve
transmission and vascular activities (Brini et al., 2013; Krebs et al., 2015). Calcium is stored in large quantity
in skeletal muscles and is the principal reservoir of calcium in the body (Burgoyne et al., 2019; Li et al., 2018). Calcium ion is essential for muscle fiber
action potential and various functions, including myosin-actin cross bridging,
protein synthesis, degradation, fiber type shifting, calcium-regulated
proteases, transcription factors, mitochondrial adaptations, plasticity, and
respiration (Gehlert et al. 2015).
Medicinal plants are known for their use in
the treatment of different
ailments because of their secondary active metabolites known to contain
numerous phytonutrients such as flavonoids, tannins, cyanogenic glycosides,
alkaloids, arocine, etc. (Agiriga & Siwela, 2017; Boy et al., 2018). Currently, active substances
extracted from higher plants are used in contemporary medicine, and 80% of
these active substances show a good association between their current
therapeutic application and their traditional uses (Prasathkumar et al., 2021). The African walnut,
Tetracarpidium conophorum, is a member of the Euphorbiaceae family.
It is an edible seed that has high nutritional content, which includes:
essential fatty acids, vitamins, and minerals. Its pharmacological effects are: antidiarrheal (Nwachoko and
Jack, 2015), pro-fertility (Dada and Aguda, 2015), antioxidant (Udedi et al.,
2014); Akomolafe et al., 2015), anti-inflammatory (Olaniyi et al., 2016), and
anti-diabetic (Ogbonna et al., 2015; Lepzem and Togun2017).Additionally, the
plant has long been used in herbal medicine to treat a variety of conditions,
including digestive and respiratory issues (Ayodeji & Aliyu, 2018). Annona muricata Lin is
a plant that grows around 4 to 8 m tall and produces green spiny heart-shaped
fruit (Chan et al., 2020). However, several pharmacological
functions have been linked to the physiochemical activities of the plants,
which include sedative, antispasmodic, hypoglycemic, hypotensive, smooth muscle
relaxant amongst others (Usunomena, 2014). Annona muricata L. is a tropical plant species known for its
edible fruit, which has some medicinal advantages and some toxicological
effects (Gavamukulya et al., 2017a).
The aim of this study was to compare the
effects of aqueous seed extract of Tetracarpidium conophorum and leaf
extract of Annona muricata on calcium level and lipid profile of male
Wistar rats.
MATERIALS
AND METHOD
Study
Area
This
Study was conducted at the Animal House, Department of Human Physiology, Faculty
of Basic Medical Sciences, College of Medicine, Chukwuemeka Odumegwu Ojukwu
University, Uli Campus.
Ethical Approval
Ethical
approval was obtained from the Faculty of Basic Medical Sciences, Chukwuemeka
Odumegwu Ojukwu University, Uli campus. Rats handling and treatments conformed
to the National Institute of Health guidelines for laboratory animal care and
use (Carbone & Austin, 2016).
METHODOLOGY
Experimental
Animals
Twenty-Five
(25) male Wistar rats weighing 150-170g were obtained from the Animal House,
Chukwuemeka Odumegwu Ojukwu University, Uli Campus, Nigeria. Animals were kept
in standard cages at a room temperature of 27±2°C. The animals were maintained
with normal laboratory chow (grower feed) and water ad libitium. The animals were acclimatized for two weeks before
administering the aqueous leaf extract of Annona
muricata and T. conophorum. The animals were kept on
12hours light and dark cycles.
Plant
Procurement and Identification
Samples
of Annona muricata (Soursop) leaves
were harvested from a local farm in Uli Community, Anambra State and seeds of
Tetracarpidium conophorum were purchased at Ihiala Market, Ihiala, Anambra
state. The two medicinal plants were identified by Dr. Bright, Department of Microbiology,
Chukwuemeka Odumegwu Ojukwu University, Uli campus, Anambra State.
Plant
Extraction Procedure
Seeds
of Tetracarpidium conophorum (walnut) and leaves of Annona muricata (Soursop) respectively were washed in running tap
water to remove dirt and air-dried under ambient temperature. The dried seeds
of Tetracarpidium conophorum and leaves of Annona muricata (Soursop) were milled into a coarse powdered form
using a local grinder, and about 250g was dissolved in 1500mls of water for
24hours. It was filtered using a clean handkerchief; after that, further
filtration was done using Whatman No. one filter paper. The filtrate was
concentrated using a rotatory evaporator, dried further using a laboratory oven
at 45°C into a gel-like form, and preserved in a refrigerator for further
usage. The extraction method was done with modifications as described according
to the method employed by Quek et al.
(2012).
Experimental Design
The
animals were randomly divided into 5 groups of
5 rats each.
Group
A: Each rat was given 1000 mg/kg aqueous seeds extract of Tetracarpidium
conophorum (T. conophorum).
Group
B: Each rat was given 500 mg/kg aqueous seeds extract of Tetracarpidium
conophorum (T. conophorum).
Group
C: Each rat was given 1000 mg/kg aqueous leaf extract of Annona muricata
(A. muricata).
Group
D: each rat was given 500 mg/kg aqueous leaf extract of Annona
muricata (A. muricata).
Group
E received feed and water ad libitum only and
served as control.
All
experimental protocols were observed under strict supervision. The experiment
lasted for 30-days, and administration was done through oral gavage.
Acute
Toxicity of Plant Extracts (Annona muricata and Tetracarpidium
conophorum)
The
median lethal dose (LD50) of the aqueous leaf extract of Annona muricata and seeds of Tetracarpidium conophorum were determined using
Lorkes method (1983). The result of acute oral toxicity
study of T. conophorum was 3807.88
mg/kg as reported by Njoku-Oji et al., 2019). That of A. muricata was 3607.52 mg/kg (Inegbenose Godwin et al., 2023).
Collection
of Blood Sample
At
the end of the experiment, animals in the different groups were anesthetized
using chloroform in an enclosed container after 24 hours of the last
administered dose of the aqueous leaf extract of Annona muricata and seed extract of Tetracarpidium conophorum.
Blood was collected from the animals through ocular puncture as described by
Parasuraman et al. (2010). Blood obtained was put in a plain
bottle. it was allowed to cool, and centrifuged for 10-minutes at 3000
revolution per minute (rpm), after which the serum was retrieved using a
micropipette. The retrieved serum was used to assay for lipid profile (total
cholesterol, low-density lipoprotein, high-density lipoprotein, triglyceride,
and very low-density lipoprotein) and calcium level.
Statistical
Analysis
The
results of this study were expressed as mean ± standard error of mean (mean ±
SEM), and data obtained were analyzed using SPSS version 21. The statistical
significance between the groups was assessed by one way analysis of variance
(ANOVA) followed by least significance difference (LSD) test with level of
significance at p<0.05.
RESULTS
Data
were analyzed using ANOVA followed by post Hoc LSD multiple comparison and
values were significant at p<0.05. (*: Significant; ns: not
significant).ASTC: aqueous seed extract of Tetracarpidium conophorum;
ALAM: aqueous leaf extract of Annona muricata.
The
result showed a non-significant increase in serum
calcium level in groups A, B, and C (p=0.718, p=0.291, p=0.643) while
group D (p=0.032) had a significant increase compared to group E.
DISCUSSION
Medicinal
plants have shown to improve lipid profile and electrolyte levels in metabolic
diseases because of the major phytochemicals they possess (Rabizadeh et al., 2022).
Lipids are important components of the cell compartments that keep life
processes intact and help regulate cardiovascular functions. However, an
abnormality of lipids can result in
complex pathological conditions such as fatty liver, cardiovascular diseases,
metabolic syndrome etc. (Cockcroft, 2021). The study investigated the effect of
aqueous seed extract of Tetracarpidium conophorum and leaf extract of Annona muricata on
calcium level and the lipid profile of male Wistar rats.
The study findings demonstrated a significant
decrease in total cholesterol level in groups A, B, and C while group D had a
non-significant decrease compared to group E. However, the physiology linked to
the significant decrease in total cholesterol level in groups treated with T.
conophorum and Annona muricata as indicated in groups A, B, and C
results from the presence of phytochemicals such as alkaloids, saponins, and
flavonoids. The study agrees with the report of Analike et al. (2017) revealing the cooked African walnuts
demonstrated a significant decline in total cholesterol level in hyperlipidemia
model, which is in accordance with the study findings. Also, Nwaichi et al. (2017) and Clarisse, Kenfack, and Angele
(2017) documented a significant reduction in total cholesterol following
administration of T. conophorum extract against hyperlipidemic
activities, which agrees with the study findings. Ezealisiji et al. (2016) reported that the ethyl acetate and
n-hexane extracts of T. conophorum seed demonstrated lowered level of
cholesterol activities against hyperlipidemic activities, which corroborates
the study outcome. Abam et al. (2013) concluded that walnut oil administered
at 2.00 g/kg and 4.00 g/kg doses restored some lipid aberrations but caused an
increase in total cholesterol, which disagree with the study findings. Also,
Ojo et al. (2022) and Florence et al. (2014) reported that aqueous extract
of A. muricata peel demonstrated a significant
reduction in total cholesterol levels in diabetic rats, which corresponds to
the study findings. However, the findings of Agu and Okolie (2019) demonstrated a significant reduction
in total cholesterol levels following Annona muricata pulp and leaf
extracts, which agrees with the study findings. Sovia et al. (2023) reported that the fruit extract of Annona
muricata demonstrated a significant reduction in total cholesterol levels
in diabetic model, which corresponds to the study findings. Adeyemi et al. (2009) and Atanu et al. (2019) reported a significant reduction in
total cholesterol level following Annona muricata leaf extract using
methanol as mode of extract against a diabetic model, which corroborates the
study findings.
Triglcerides (TGs) are important in the
metabolism of fatty acids through the de novo biosynthesis. Fatty acids are
eliminated by oxidation within the cell or by secretion into the plasma within
triglyceride-rich very low-density lipoproteins. However, TGs are associated
with non-alcoholic fatty liver, which
results from obesity, type 2 diabetes, and dyslipidemia, and commonly occurs in
the setting of insulin resistance (Alves-Bezerra & Cohen, 2017). The triglyceride result revealed a
significant reduction in groups A and C while groups B and D had a
non-significant decrease compared to group E. The mechanism of action following
the reduction in the TGs level is associated with flavonoids and alkaloids
present in both extracts, which tends to lower cholesterol through
esterification process. The reports of Analike et al. (2017), Nwaichi et al.
(2017), Ezealisiji et al. (2016), and Abam et al. (2013) showed similarity to
the study findings documenting a significant reduction in TGs levels following
administration of T. conophorum against toxicity of different models.
However, treatments with Annona muricata demonstrated a lowered level of
TG, which could be attributed to the presence of flavonoids in the plant. The
study of Ojo et al. (2022), Florence et al. (2014), Atanu et al. (2019), and
Sovia et al. (2023), reported a significant reduction in TGs levels following
administration of Annona muricata, which agree with the study findings.
Lipoproteins, lipid molecules involved in
transporting proteins and lipid metabolism, are a risk factor for
cardiovascular disease (Bhargava et al., 2022). Genetic and environmental disorders
can lead to abnormal levels or functions of lipoproteins, increasing the risk
of cardiovascular disease. Factors like diet, physical activity, and smoking
also influence lipoprotein levels, contributing to the development of lipid
disorders (Lent-Schochet & Jialal, 2023). The study findings showed a
non-significant decrease in the LDL-C levels in groups A and D while groups B
and C had a significant decrease compared to group E. The HDL-C result showed a
non-significant increase in groups A, B, C, and D compared to group E. The
mechanism of action linked to decreased level of LDL-C in groups B and C is associated
with flavonoids, alkaloids, and saponins present in T. conophorum and Annona
muricata.
The findings of Analike et al. (2017) showed
significant decrease in LDL following administration of cooked walnuts, which
agrees with the study findings. Also, Nwaichi et al. (2017) demonstrated a
significant reduction in LDL following T. conophorum against
hyperlipidemia toxicity, which corroborates the study result. Further, the
ethyl acetate and n-hexane extracts of T. conophorum seed demonstrated a
significant decline in LDL-cholesterol levels, which agree with the study
findings (Ezealisiji et al., 2016). Further, the oil of T. conophorum demonstrated
a significant reduction in LDL-cholesterol levels, which agrees with the study
findings. Talabi et al. (2023) showed a lowered level of LDL-cholesterol
following T. conophorum, which agrees with the study findings. Further,
treatments with Annona muricata from several studies (Agu and Okolie,
2019; Florence et al., 2014, Sovia et al., 2023) indicated a significant
reduction in LDL-cholesterol levels in toxicity studies, which agree with this
study outcome. However, the study disagrees with the report of Florence et al.
(2014) demonstrating a significant increase in HDL-cholesterol level following Annona
muricata ingestion against diabetic model. Also, Agu and Okolie (2019)
reports contradict the study findings demonstrating a significant increase in
HDL-cholesterol level following Annona muricata in hyperlipidemic
activities. Clarisse, Kenfack, and Angele (2017) showed a significant increase
in the HDL levels following T. conophorum intake and contradict the
study outcome. Ezealisiji et al. (2016) indicated a significant increase in the
HDL levels following T. conophorum intake, which refutes the study
outcome.
Calcium ion is the most significant ion in
the heart, which is known for its function in the contraction of cardiac
muscle. However, calcium dysfunction has been linked to the onset of cardiac
muscular dystrophy (Agrawal et al., 2018). The study showed a non-significant
increase in serum calcium level in groups A, B, and C while group D had a
significant increase compared to group E. The mechanism of action following the
significant increase in calcium level is not well understood but it could be
suggested that Annona muricata contains minerals (calcium), which tends
to replenish the depleted calcium stores in the body that might affect the
cardiac and skeletal muscle function. However, the findings of Elizabeth et al.
(2018) showed an increase in the calcium
level when the root and leaf extract of Annona muricata were compared,
which agrees with the study findings.
CONCLUSION
The
study revealed that T. conophorum and Annona muricata extracts
significantly reduced cholesterol, triglycerides, and low-density lipoprotein cholesterol
and non-significantly increased HDL-C. Annona muricata alone
significantly increased calcium level.
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