INTRODUCTION
Cardiovascular
diseases (CVDs) remain a leading cause of mortality worldwide, with
dyslipidemia—abnormal levels of lipids in the blood—serving as a major risk
factor. Elevated cholesterol and triglycerides accelerate the development of
atherosclerosis, a key contributor to heart attacks and strokes (Giacco & Brownlee, 2010). Diabetes mellitus further
exacerbates this risk, as hyperglycemia is often accompanied by dyslipidemia,
which together enhance oxidative stress and lipid peroxidation (Madamanchi, Vendrov, & Runge, 2005). Oxidative stress, defined as an imbalance
between reactive oxygen species (ROS) and the body's antioxidant defenses, is a
major factor in both the onset and progression of diabetes-related
complications, including cardiovascular disease (Giacco
& Brownlee, 2010).
Pharmacological
interventions for managing dyslipidemia and diabetes, such as statins and oral hypoglycemics, are widely used but are often accompanied by
adverse effects, including muscle pain, liver dysfunction, and gastrointestinal
disturbances (Thompson, Clarkson, & Karas, 2003).
These challenges have fueled interest in exploring natural alternatives,
particularly plant-based remedies, which offer potential for managing metabolic
disorders with fewer side effects.
Costus lucanucianus, a medicinal plant
traditionally used in African medicine, is known for its anti-inflammatory,
antihypertensive, and gastrointestinal healing properties (Iwu,
1993). Phytochemical studies indicate that the plant is rich in bioactive
compounds, such as polyphenols and flavonoids, which exhibit antioxidant and
lipid-lowering effects (Mujeeb, Bajpai,
& Pathak, 2014). However, the plant’s potential to mitigate the combined
effects of diabetes and dyslipidemia, particularly in relation to oxidative
stress and lipid metabolism, remains largely unexplored.
This
study aims to evaluate the effects of the stem extract of Costus
lucanucianus on lipid profile and oxidative
stress markers in diabetic and dyslipidaemic male Wistar rats. Specifically, the study assessed the plant's
impact on serum lipid levels—including total cholesterol (TC), triglycerides
(TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein
cholesterol (LDL-C)—as well as oxidative stress indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase
(CAT), and glutathione peroxidase (GPx). The goal is
to investigate the therapeutic potential of C. lucanucianus
in managing metabolic disturbances associated with both diabete-s
and dyslipidemia.
MATERIALS AND METHODS
Plant Material and Extraction
Mature stems of Costus
lucanucianus were harvested from the forest
reserve in Biara community, located in Gokana Local Government Area, Rivers State, Nigeria. The
plant material was identified and authenticated by a taxonomist in the
Department of Plant Science and Biotechnology, Rivers State University, and a
voucher specimen (Herbarium number: RSUPbO104) was deposited in the
institution’s herbarium for future reference.
Following
collection, the stems were thoroughly washed with clean water to remove dirt
and other contaminants. The cleaned stems were air-dried at room temperature
(32–35°C) for a period of 21 days until constant weight was achieved. The dried
stems were then ground into a fine powder using a mechanical grinder, and the
pulverized sample was sieved to ensure uniform particle size. The resulting
fine powder was stored in a sterile, air-tight container to prevent moisture
absorption or contamination, until required for extraction.
For the extraction
process, 250 g of the pulverized stem sample was weighed and subjected to cold maceration
using 2.5 L of 99% ethanol as the extraction solvent. The mixture was allowed
to macerate for 72 hours at room temperature with occasional stirring and
agitation to enhance the extraction of bioactive compounds. After 72 hours, the
mixture was filtered through Whatman No. 1 filter
paper to obtain the filtrate, and the marc (residue) was discarded.
The ethanol filtrate
was concentrated to dryness under reduced pressure using a rotary evaporator at
a temperature of 45–50°C. This process removed the ethanol, yielding a
semi-solid crude extract. The dried extract was collected, weighed, and the
percentage yield was calculated using the following formula:
% of extractive yield (w/w) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑖𝑒𝑑 𝑒𝑥𝑡𝑟𝑎𝑐t x 100
𝑊𝑒𝑖𝑔ℎ𝑡𝑜𝑓𝑑𝑟𝑖𝑒𝑑 𝑠𝑡𝑒𝑚 𝑝𝑜𝑤𝑑𝑒𝑟
The resulting semi-solid extract was
stored in pre-weighed, sterile screw-capped bottles, labeled accordingly, and
kept refrigerated at 4°C to maintain its stability and prevent degradation
until further analysis.
Experimental Animals
Thirty (30) healthy
male Wistar rats, with body weights ranging from 110
to 250 g, were obtained from Rogers Farm, Afam,
located in Oyigbo Local Government Area of Rivers
State, Nigeria. The animals were housed in clean, well-ventilated cages within
the animal facility of the Faculty of Basic Medical Sciences. They were
provided with a standard laboratory diet and clean drinking water ad libitum
throughout the duration of the study. Bedding, water, and cages were maintained
in a clean condition through daily sanitation procedures.
The study was
conducted in accordance with the guidelines set forth by the Institutional
Animal Ethics Committee, ensuring adherence to ethical standards for the use of
animals in research.
During the
experimental period, twenty (20) of the rats were placed in a controlled
environment with a standard high-fat diet and fresh drinking water for a
duration of five (5) weeks to induce hyperlipidemia. The remaining ten (10)
rats continued to receive a normal diet, serving as the control group. All
animals were maintained under a natural light/dark cycle of 12 hours each,
providing a stable environmental condition for the duration of the experiment.
Hyperlipidemia was
assessed using the Vivadiag lipid testing system,
with blood samples collected from the tail vein of the rats. This method
facilitated the measurement of serum lipid profiles to confirm the induction of
hyperlipidemia in the experimental group.
Induction of Diabetes and Dyslipidemia
Diabetes mellitus was induced in overnight-fasted
experimental rats through a single intraperitoneal
injection of Alloxan monohydrate at a dose of 150
mg/kg body weight, as per the methods described by Onyebuchi
et al. (2016) and Dewanjee et al. (2008). The alloxan monohydrate was dissolved in 0.9% normal saline
prior to administration.
To mitigate the risk
of hypoglycemic shock following the induction of diabetes, the rats were
provided with a 5% glucose solution as their drinking water for the first 12
hours post-injection. Three days after the administration of alloxan, blood glucose levels were confirmed using an
On-Call Plus glucometer, with blood samples obtained via tail puncture.
Rats exhibiting
fasting blood glucose levels greater than 200 mg/dL
were classified as diabetic. In addition to hyperglycemia, lipid profiles were
assessed, with total cholesterol levels exceeding 110 mg/dL
and triglyceride levels greater than 150 mg/dL used
as criteria for dyslipidemia. Rats meeting these lipid profile criteria were
selected for inclusion in the study.
Experimental Design
The experimental animals were randomly
divided into five groups, with each group consisting of five rats, as outlined
below:
Group I: Normal control group. Rats in
this group received a standard diet and distilled water.
Group II: Diabetic-dyslipidaemic
control group. Rats in this group were fed a normal diet and provided with
distilled water.
Group III: Treatment group. Diabetic-dyslipidaemic rats received 250 mg/kg body weight of
Metformin and 10 mg/kg body weight of Atorvastatin.
Group IV: Treatment group. Diabetic-dyslipidaemic rats were administered 200 mg/kg body weight
of Costus lucanucianus
extract.
Group V: Treatment group. Diabetic-dyslipidaemic
rats received 400 mg/kg body weight of Costus
lucanucianus extract.
Study Procedure
Before the
initiation of extract administration, the body weights of all rats were
measured using a digital animal weighing scale. These measurements were
recorded weekly throughout the study. Additionally, lipid profile assessments
were performed weekly using the Vivadiag lipid
testing system, with results meticulously documented. The site of tail puncture
was disinfected with normal saline before each blood sampling to minimize the
risk of contamination.
Induction of Dyslipidaemia
Dyslipidaemia was induced by administering
a high-fat diet, which included Blue Band margarine. Specifically, 45 g of
margarine, containing 84% saturated fat, was mixed with the standard diet of
the animals daily over a period of five weeks. Weekly lipid profile analyses
were conducted to monitor serum lipid levels until dyslipidaemia
was confirmed.
Stock Solution Preparation
The stock
solution of the extract was prepared by dissolving the semi-solid extract in 35
mL of dimethyl sulfoxide (DMSO), following the
methodology outlined by Erhirhie et al. (2014).
Extract Administration
Following
the preparation of the stock solution, the extract was administered to the rats
based on their individual body weights. Oral administration was conducted using
a gavage tube and a 2 mL syringe to ensure precise dosing.
Collection of Blood Samples and Tissues
The rats
were treated for a duration of twenty-one (21) days. At the conclusion of the
treatment, blood glucose levels were measured using a glucometer, and lipid
profiles were analyzed using the Vivadiag lipid
testing system. Blood samples were collected from the tail vein via puncture.
Following the treatment period, the animals were subjected to a 6-hour fasting
period. Blood samples were subsequently obtained through cardiac puncture after
inducing sedation with chloroform. Serum was separated from the red blood cells
using a centrifuge. Laparotomy was performed to extract the liver and pancreas
for histopathological examination.
Biochemical Analysis
- Lipid Profile: The lipid
profile analysis was conducted using the Vivadiag
lipid testing system, utilizing venous blood samples obtained via tail
puncture from the rats. The instrument automatically measured and recorded
the levels of total cholesterol, triglycerides, high-density lipoprotein
(HDL), and low-density lipoprotein (LDL) in the collected blood samples.
- Oxidative Stress Markers: The serum levels of catalase,
superoxide dismutase (SOD), glutathione peroxidase, and malondialdehyde (MDA) were quantified using
established methodologies. Specifically, catalase activity was measured
following the method described by Aebi (1974),
while superoxide dismutase activity was assessed using the protocol
outlined by Fredovich (1989). The levels of
glutathione peroxidase and malondialdehyde were
determined according to the procedures established by Ellman
(1959).
Statistical Analysis
Statistical analysis
was done using Statistical Package for Social Sciences (SPSS) version 21. Data
were expressed as mean ± SEM. Values were compared using analysis of variance
(ANOVA) and differences in the values were considered statistically significant
when p<0.05. Results were presented
on tables and graph.
RESULTS
The phytochemistry of the Costus lucanusianus was analyzed and is shown
in Table 1. The Alkaloid, phenolic constituents, anthraquinone,
cardenolide were reportedly absent. Salwoski test performed for triterpenoid
showed that it was present. Fixed oil, carbohydrates, cyanogenic
glycosides and saponins were all present.
DISCUSSION
The
phytochemical analysis of the stem of Costus
lucanusianus (Table 1) revealed the presence of triterpenoids, fixed oils, carbohydrates, cyanogenic glycosides, and saponins,
while alkaloids, phenolic compounds (including flavonoids), anthraquinones,
and cardenolides were absent. Triterpenoids,
known for their anti-inflammatory, antioxidant, and hypolipidemic
properties, are likely contributors to the plant's effects on lipid metabolism
and oxidative stress. Studies such as Sun et
al. (2017) have shown that triterpenoids can
reduce hyperglycemia and improve lipid profiles in diabetic models, supporting
their potential role in this study. Saponins, known
for their cholesterol-lowering properties, could explain the observed decrease
in LDL and total cholesterol levels. Despite the absence of alkaloids and
phenolic compounds, Costus lucanusianus demonstrated significant antioxidant
effects, which may be attributed to the combined action of triterpenoids
and saponins.
The
effects of the ethanol stem extract of Costus
lucanusianus on the serum lipid profile of Wistar rats, as presented in the results, show promising
lipid-modulating properties. The study demonstrates significant reductions in
total cholesterol, triglycerides, and low-density lipoprotein (LDL) levels in
the treated groups, particularly with the 400 mg/kg dose of the extract. In
contrast, the positive control group (untreated diabetic-dyslipidemic
rats) exhibited the highest levels of these lipid parameters, indicating the
severity of dyslipidemia in the absence of treatment.
In
the 400 mg/kg treatment group, total cholesterol levels were significantly
reduced to 110.81±1.51 mg/dl, which was notably lower than the positive control
group's 182.28±2.27 mg/dl (p<0.05). This substantial reduction suggests that
Costus lucanusianus has
a potent cholesterol-lowering effect. Similar findings have been observed in
other medicinal plants known for their hypocholesterolemic
properties. For example, a study on Gynura procumbens demonstrated a significant reduction in total
cholesterol in diabetic rats, which aligns with the cholesterol-lowering
effects seen with Costus lucanusianus extract (Mohan et al., 2013). The cholesterol-lowering mechanism may be attributed
to the phytochemicals present in the extract, such as saponins,
which are known to inhibit cholesterol absorption in the intestines (Sarkar
& Choudhury, 2016).
The
reduction in triglycerides and LDL levels in the 400 mg/kg group was also
significant (p<0.05), with values of 116.29±0.92 mg/dl for triglycerides and
139.17±3.3 mg/dl for LDL. These findings are consistent with previous research
that highlights the importance of lipid management in diabetic conditions,
where elevated triglycerides and LDL levels contribute to cardiovascular risk (Friedewald et al.,
1972). A similar study on Moringa oleifera
extract also demonstrated a significant reduction in triglycerides and LDL
levels in diabetic rats, likely through its antioxidant and anti-inflammatory
properties (Fahey, 2005).
In
the positive control group, which had the highest values for triglycerides
(298.72±2.74 mg/dl) and LDL (187.37±2.20 mg/dl), the absence of treatment
underscores the dyslipidemic effects of diabetes, as dysregulated lipid metabolism is a hallmark of the disease
(American Diabetes Association, 2020). The ability of the Costus
lucanusianus stem extract to counter these
effects suggests its potential as an adjunct therapy for managing dyslipidemia
in diabetic patients.
Interestingly,
Costus lucanusianus
also increased HDL levels significantly (p<0.05). The 200 mg/kg and 400
mg/kg groups recorded HDL values of 50.38±3.04 mg/dl
and 51.22±1.83 mg/dl, respectively, compared to 30.20±3.28 mg/dl in the
positive control group. HDL is known for its cardioprotective
role, as it helps transport cholesterol from peripheral tissues back to the
liver for excretion (Gordon et al.,
1977). The increase in HDL levels induced by Costus
lucanusianus is comparable to the effects of
other hypolipidemic agents. For instance, research on
Cinnamomum zeylanicum
extract showed similar HDL-boosting effects in diabetic rats (Khan et al., 2003).
Furthermore,
the standard drug group (treated with metformin and lovastatin) recorded the
highest HDL value of 58.15±15 mg/dl, reinforcing the idea that standard
lipid-lowering medications remain highly effective. However, the HDL increase
in the Costus lucanusianus
treated groups suggests that this plant extract might offer a natural
alternative with fewer side effects, an advantage supported by studies on
plant-based lipid modulators (Gotto, 1990).
Other
research supports the findings of this study. For example, Allium sativum (garlic) has been reported to lower cholesterol and
triglyceride levels while increasing HDL in diabetic models, similar to the
effects of Costus lucanusianus
(Silagy & Neil, 1994). Additionally, polyphenolic compounds in plants like Costus lucanusianus are known
to improve lipid profiles by enhancing the activity of enzymes involved in
lipid metabolism and reducing oxidative stress, which contributes to lipid dysregulation (Bhattacharya, 2005).
The
results in Table 3 demonstrate the effects of the ethanol extract of Costus lucanusianus
on oxidative stress markers in Wistar rats, showing
its potential antioxidative properties. Oxidative
stress markers, including glutathione (GSH), catalase (CAT), superoxide
dismutase (SOD), and malondialdehyde (MDA), were
significantly altered in response to different doses of the extract, indicating
its role in modulating oxidative stress.
Glutathione
is a key antioxidant that plays a vital role in neutralizing free radicals and
protecting cells from oxidative damage. The group treated with 200 mg/kg of Costus lucanusianus
stem extract recorded the lowest GSH value of 0.88±0.12 µg/ml, which was
significantly lower (p<0.05) than the negative control group (1.93±0.38
µg/ml). Similarly, the 400 mg/kg treatment group showed a GSH level of
1.27±0.18 µg/ml, which, while lower than the control group, was higher than the
200 mg/kg treatment group, suggesting a dose-dependent increase in glutathione
levels.
A
similar trend has been observed in other studies where plant extracts rich in
antioxidants improve the endogenous levels of glutathione in oxidative stress
models. For instance, the aqueous extract of Moringa oleifera demonstrated a significant increase in GSH levels
in diabetic rats, comparable to the effects seen in the 400 mg/kg treatment
group in this study (Sreelatha & Padma, 2009).
Catalase
is another crucial antioxidant enzyme that protects cells from oxidative damage
by catalyzing the decomposition of hydrogen peroxide into water and oxygen. In
this study, the group treated with 200 mg/kg of the extract recorded a catalase
activity of 2.60±0.46 µg/g, significantly lower than the negative control
(5.18±0.33 µg/ml) (p<0.05). However, the group treated with 400 mg/kg of the
extract showed an improved catalase activity of 3.07±0.57 µg/ml, indicating
that the higher dose of the extract provided better protection against
oxidative stress.
These
results are in line with findings from research on other medicinal plants, such
as Curcuma longa (turmeric), which has been shown to significantly increase
catalase activity in diabetic rats due to its strong antioxidant properties (Akinmoladun et al.,
2010).
Superoxide
dismutase (SOD) is an enzyme responsible for the dismutation
of superoxide radicals into oxygen and hydrogen peroxide, playing a crucial
role in reducing oxidative stress. The 200 mg/kg extract-treated group recorded
a significantly lower SOD activity of 0.20±0.03 µg/ml compared to the negative
control group (0.43±0.03 µg/ml) (p<0.05). However, the group treated with
400 mg/kg of the extract had an improved SOD level of 0.38±0.02 µg/ml,
indicating that the higher dose enhanced the rats' antioxidant defense.
SOD
activity is frequently used as a marker of oxidative stress in experimental
models, and various plant extracts have demonstrated similar effects in
increasing SOD levels. A study on Phyllanthus amarus showed that its administration significantly
improved SOD levels in oxidative stress-induced conditions, a trend consistent
with the effects seen with Costus lucanusianus extract (Prakash et al., 2011).
Malondialdehyde (MDA) is a
marker of lipid peroxidation, which indicates the extent of oxidative damage to
cell membranes. In this study, the group treated with 200 mg/kg of Costus lucanusianus
recorded an MDA level of 0.59±0.02 µmol/ml,
significantly lower than the negative control group (0.27±0.44 µg/ml)
(p<0.05). The group treated with 400 mg/kg of the extract had an even lower
MDA value of 0.41±0.04 µg/ml, indicating that the extract effectively reduced
lipid peroxidation and oxidative damage, with the higher dose providing more
protection.
These
results are in agreement with other studies that have shown the ability of
plant extracts to reduce MDA levels in oxidative stress models. For example, Vernonia amygdalina was found to
significantly reduce MDA levels in diabetic rats, similar to the results seen
with Costus lucanusianus
extract (Oboh et
al., 2014).
CONCLUSION
The
study indicates that the ethanol extract of Costus
lucanusianus exerts significant lipid-modulating
and antioxidant effects in diabetic-dyslipidemic Wistar rats. The plant's phytochemical constituents,
especially triterpenoids and saponins,
appear to play crucial roles in reducing cholesterol, triglycerides, and LDL levels,
while simultaneously enhancing HDL levels. Additionally, the extract
effectively mitigated oxidative stress, as evidenced by improved levels of GSH,
CAT, SOD, and reduced MDA. These findings support the potential use of Costus lucanusianus
as a natural therapeutic agent for managing dyslipidemia and oxidative stress
in diabetic conditions.
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