ABSTRACT
The effects of methanol leaf extracts of Telfairia occidentalis. and Amaranthus hybridus on aspartame-induced oxidative stress in wistar Albino rats was investigated. Rats were randomly selected and divided into 5 groups (5 rats per group) and acclimatized for two weeks. Group 1 was the normal group. Group II (negative control) was given aspartame (40 mg/kg b.w) for 40 days. Groups III and IV were also given aspartame but treated with 200 mg/kg b.w methanol leaf extracts of Amaranthus hybridus and Telfairia occidentalis respectively. Group V was also given aspartame but subjected to co-administration of 100 mg/kg b.w each of both methanol leaf extracts of Amaranthus hybridus and Telfairia occidentalis in the ratio of 1:1 for 40 days. All the rats were fed with commercial rat chow and water ad libitum throughout the experimental period. They were then sacrificed after an overnight fast and blood collected for determination of various parameters. The results revealed a significant increase (p<0.05) in malondialdehyde (MDA) concentration in Group II (negative control) compared to the normal and treated groups III and IV but Group V recorded a significant decrease (p<0.05) in the MDA concentration compared to the other treated groups and the normal. The activities of the antioxidant enzymes superoxide dismutase and catalase were significantly decreased (p<0.05) in Group II (negative control) compared to the normal and the treated groups though catalase activity was significantly lower (p< 0.05) in the normal compared to the other treated groups. Total cholesterol concentration was significantly increased (p<0.05) in Group IV compared to other test groups and the normal while there was a significant decrease (p<0.05) in Group V compared to the normal. LDL-cholesterol concentration in Group II (negative control) recorded a significantly high increase (p<0.05) compared to all the other groups and the normal. There were no significant changes in triacylglycerols (TAGs), HDL-cholesterol and VLDL (p>0.05) in all the groups compared to the normal. Liver enzymes activity showed no significant change in AST (p>0.05) while ALT was significantly decreased (p<0.05) in all the groups compared to the normal. ALP was significantly decreased (p<0.05) in all the treated groups and the negative control compared to the normal. Total bilirubin concentration was significantly increased (p<0.05) in group V compared to the normal and the other test groups. Direct bilirubin was significantly decreased (p<0.05) in Group II and in Group III compared to the normal and the other treated groups. Total protein and globulins were significantly increased (p<0.05) in all the treated groups compared to the normal and the negative control. Albumin concentration showed no significant change (p>0.05) in all the groups compared to the normal. Serum creatinine and urea concentrations recorded no significant change (p>0.05) in all the treated groups and in the negative control group compared to the normal. These results suggest that aspartame has oxidative properties as evidenced by the significantly increased MDA concentration and decreased activities of the antioxidant enzymes in Group II (negative control) compared to the normal and the treated groups while Amaranthus hybridus and Telfairia occidentalis may have antioxidant capacities that can ameliorate oxidative stress.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables x
List of Plates xi
Abstract xii
CHAPTER 1:
INTRODUCTION
1.1 Background
of the Study 1
1.2 Statement
of the Problem 4
1.3 Aim of
the Study 4
1.4 Objectives
of the Study 4
1.5 Scope 5
1.6 Significance
of the Study 5
1.7 Justification
of the Study 5
CHAPTER 2:
LITERATURE REVIEW
2.1 Description
of Telfairia occidentalis 6
2.1.1 Scientific
classification of Telfairia occidentalis 7
2.1.2 Structure,
cultivation and storage 8
2.1.3 Nutritional
potentials of Telfairia occidentalis 9
2.1.4 Medicinal
properties/health benefits of Telfairia
occidentalis 11
2.2 Description
of Amaranthus hybridus 14
2.2.1 Scientific
classification of Amaranthus hybridus 15
2.2.2 Distribution 16
2.2.3 Nutritional
potentials of Amaranthus hybridus 17
2.2.4 Medicinal
properties/health benefits of Amaranthus
hybridus 17
2.3 Aspartame 21
2.3.1 Chemistry
and mechanism of action 21
2.3.2 Uses of
aspartame 22
2.3.3 Safety
and health effects 23
2.4 Oxidative
Stress 24
2.4.1 Causes
of oxidative stress 24
2.4.2 Oxidants
and their sources 26
2.5 Lipid
Profile 28
2.6 Liver
Function 29
2.6.1 Liver
function and enzyme markers 29
2.6.2 Plasma
and serum proteins 32
2.6.3 Bilirubin 32
2.7 Kidney
Function and Kidney Function Tests 32
CHAPTER 3:
MATERIALS AND METHODS
3.1 Materials 34
3.1.1 Plant
materials sampling 34
3.1.2 List of
chemicals used 34
3.1.3 List of
apparatus and instruments 35
3.2 Methods 36
3.2.1 Extraction
of plant materials 36
3.2.2 Experimental
design and animal grouping 36
3.2.3 Collection
of blood and preparation of serum samples 37
3.3 Biochemical
Assay 37
3.3.1 Assay
for lipid peroxidation (malondialdehyde) 37
3.3.2 Antioxidant
assays 38
3.3.2.1 Determination
of catalase activity 38
3.3.2.2
Determination of superoxide dismutase (SOD) activity 39
3.3.3 Determination
of serum lipid profile 40
3.3.3.1 Determination of total cholesterol (TC) 40
3.3.3.2
Determination of triacylglycerols (TAGs) 41
3.3.3.3
Determination of HDL – cholesterol 42
3.3.3.4
Estimation of LDL– cholesterol 42
3.3.3.5
Estimation of VLDL 42
3.3.4 Analyses
of liver function 43
3.3.4.1
Determination of serum aspartate amino transferase (AST) activity 43
3.3.4.2 Determination
of serum alanine amino transferase (ALT) activity 44
3.3.4.3 Determination
of serum alkaline phosphatase (ALP) activity 44
3.3.4.4 Determination
of serum bilirubin fractions 45
3.3.4.4.1 Determination of total bilirubin 45
3.3.4.4.2 Determination of direct bilirubin 46
3.3.5 Analyses
of proteins 46
3.3.5.1 Determination
of serum total proteins (TP) 46
3.3.5.2 Determination
of serum albumin 47
3.3.5.3 Determination
of globulins 48
3.3.6 Determination
of kidney function parameters 48
3.3.6.1 Determination
of serum creatinine 48
3.3.6.2 Determination
of serum urea 49
3.4 Statistical Analysis 50
CHAPTER 4: RESULTS
AND DISCUSSION
4.1 Results 51
4.2 Discussion 57
CHAPTER 5:
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion 65
5.2 Recommendations 65
References 66
Appendices 76
LIST OF TABLES
Page
4.1: Effects
of Amaranthus hybridus and Telfairia
occidentalis methanol
leaf
extracts on malonaldehyde concentration (lipid peroxidation marker)
in
aspartame-treated rats 51
4.2:
Effects of Amaranthus hybridus and Telfairia
occidentalis methanol leaf
extracts
on some antioxidant enzymes in aspartame –treated rats.- 52
4.3 Effects
of Amaranthus hybridus and Telfairia
occidentalis methanol
leaf
extracts on the lipid profile of aspartame –treated rats 53
4.4 Effect
of of Amaranthus hybridus and Telfairia
occidentalis methanol
leaf extracts on liver function of aspartame – treated rats. 54
4.4 (a) Activities of liver enzymes
4.4
(b) Effects of Amaranthus hybridus and Telfairia
occidentalis methanol
leaf extracts on liver function of
aspartame – treated rats 55
4.4(c) Effects of Amaranthus hybridus and Telfairia
occidentalis methanol
leaf extracts on other parameters of
liver function of aspartame-treated rats 56
4.5: Effects of Amaranthus hybridus and Telfairia
occidentalis methanol leaf extracts
on some kidney functions parameters of aspartame –treated rats 57
LIST OF PLATES
2.1: Plate showing young shoots, leaves and
fruit of Telfairia occidentalis 8
2.2: Plate showing the leaves, stems and seeds
of Amaranthus hybridus 16
CHAPTER 1
INTRODUCTION
1.1
BACKGROUND OF THE STUDY
Green
leafy vegetables are an essential component of the foods consumed by animals
and humans. Biblically the foods given to man by God and animals after creation
were basically foods of plant origin. It is believed that God gave man the
permission to eat foods of animal origin because plants had not yet begun to
grow and yield fruits immediately after the flood, and so only foods derived
from the animals in Noah’s ark were available for immediate consumption
(Malkmus et al., 2006).
A
diet consisting mainly of vegetables, fruits, whole grain cereals rich in
unrefined carbohydrate and dietary fibre is best for maintenance of good health
because most plants generally have good medicinal properties. This is
principally because plants contain a wide variety of vitamins and phytochemicals
which promote good health. Also, the dietary fibre present in plants, in whole
grain cereals and unrefined carbohydrate further contribute to good health and
protects against disease. Together with the phytochemicals such as flavonoids
and the antioxidant vitamins like vitamin C and E, plant-based food products
can act as scavengers of oxygen- derived free radicals (Rolfe et al., 2006) which are generated
inherently from normal metabolic reactions in the body, and also in part by
environmental factors such as pesticide residues in foods and ionizing
radiation which one cannot help being exposed to due to increasing
industrialization and the rural –urban drift. The need to include more
vegetables in our diet so as to maintain good health is gaining increasing
importance in recent times. The enormous benefits of eating more fruits and
vegetables on a daily basis have been unraveled by many researchers (Rolfe et al.,2006; Kayode and Kayode, 2011;
Usunobun and Egharebva, 2014). Plants not only provide the nutrients needed for
growth and development, they also provide phytochemicals against many diseases.
The art of using plants for medicinal purposes is as old as man himself and the
search for medicinal plants is unending because of the many advantages they confer.
The
increasing use of food additives, chemical preservatives and food flavor
enhancers is a major health concern. Aspartame is an artificial sweetener used
in food products as a sugar alternative. Its sweetness is about 200 times more
than that of sugar thus it is used in low-calorie soft drinks and foods.
Aspartame was discovered serendipitously in 1965 by researchers trying to come
up with a new anti-ulcer drug. It was approved by the Food Drug Administration
(FDA) In 1981 to be used in dry applications and then certified for use in
carbonated beverages in 1983 and as a general sweetener in 1986 (Butchko et al., 2002). When ingested, aspartame
is hydrolyzed by digestive esterases and peptidases in the intestinal lumen to
methanol and the amino acids phenylalanine and aspartate and this increases the
concentration of these metabolites in the blood (Stegink et al., 1987). A panel of experts in 2013 concluded that at current
level of exposure, aspartame is safe for human consumption (El-bahr, 2013). Up till
2017, evidence does not support a long term benefit for weight loss or for
diabetics (Azad et al., 2017). People
with the inborn error, phenylketonuria (PKU), must be careful of products which
contain aspartame because of phenylalanine which is one of its breakdown
products.
Even
though aspartame produces 4 kcal of energy per gram (17 KJ/g) when metabolized,
its caloric contribution is negligible because only very minute quantity is
needed for it to produce sweetness (Magnuson et al., 2007). The sweetness of aspartame outlasts that of sucrose
and so it is usually blended with other artificial sweeteners such as
acesulfame potassium to produce an overall taste like that of sugar (O’Donnel,
2006).
Since being approved as an artificial
sweetener in 1981, aspartame has been the subject of much controversy regarding
its effects on health. There have been concerns that aspartame might be
carcinogenic (Olney, 1996). Reports reveal that aspartame causes headaches,
seizures and mood changes (Magnuson et al.,
2007). However, these claims have been disputed by other researchers. The
toxicity of aspartame has been linked to its pro-oxidative effects in animal
studies.
Most
diseases such as cancer, asthma, cardiovascular diseases, diabetes mellitus,
arthritis etc, and aging are associated with oxidative stress (Rolfe et al., 2006, Murray, 2006; Chitanya et al., 2010). Oxidative stress results
when there is excessive production of oxygen- derived free radicals in cells.
This could overpower the normal antioxidant capacity of the cells to detoxify free
radicals. When the concentration of oxygen-derived free radicals is more than
the antioxidants species such as tocopherols, ascorbic acid and glutathione or
enzymes involved in oxygen radical scavenging (catalase, peroxidases, and
superoxide dismutase (SOD), oxidative damage occurs to proteins, lipids and
DNA, leading to cytotoxicity, mutation and carcinogenesis (Murray, 2006;
El-bahr, 2013).
Telfairia occidentalis is a creeping vegetable plant widely
cultivated in West Africa for its succulent leaves and edible seeds. Common names for the plant include fluted
gourd; fluted pumpkin; “Ugu” and “Ikong Ubong” in the Igbo and Efik/Ibibio
languages respectively. It is a member of the Curcubitaceae family, and is
native to Southern Nigeria. It is highly coveted because of its high nutritive
value and healing properties. Interest is constantly increasing in the use of
this vegetable to combat hepatotoxicity (Kayode et al., 2011), because the leaves and young shoots are rich in phytochemicals
and antioxidants (Adaramoye et al.,2007;
Kayode et al., 2009; Kayode et
al., 2010).
Several authors have reported the ability of Telfairia occidentalis in protecting
against liver damage (Oboh et al., 2005).
Amaranthus hybridus, commonly called green amaranth is a herbal plant usually grown for
consumption. It is named “Terere” in
most communities in Kenya and is cultivated in various areas of the world such
as South America, Africa, India, China and the United States (Kavita and Ghandi
, 2002). This vegetable is a known important source of iron. The leaves of this
plant is said to have many medicinal properties; tea produced from the leaves
of Amaranthus hybridus is astringent
and is used for treating intestinal bleeding, diarrhea, and excessive
menstruation (Foster and Duke, 1990).
Oxidative
stress is the underlying cause of many debilitating diseases and this has been
well documented in the literature (Murray, 2006; Friedovich, 2007). It is also
a fact that plant-based diets and leafy vegetables generally have beneficial
effects on health. It is also worthy of note that no matter how harmless
synthetic food additives may be, the body still recognizes them as foreign
compounds and this may result in reactions which may be injurious to health
(Malkmus et al., 2006). This study
therefore sought to explore the potentials of these two plants – Telfairia occidentalis and Amaranthus hybridus against the effect
of aspartame, by investigating numerous biochemical parameters which may give
an insight into the health effect of aspartame vis-à-vis the opposing and/or
interactive effects of these plants.
1.2 STATEMENT OF THE
PROBLEM
There
is an increasing use of aspartame in mineral drinks, candies, pharmaceuticals
etc. in place of sucrose. It is therefore necessary to study the effect of this
sweetener on the body, and to investigate the ability of commonly eaten
vegetables to ameliorate such effects. (Magnuson et al., 2007; Oboh et al.,
2006; Kayode et al., 2010).
1.3 AIM OF THE STUDY
The
aim of this study was to investigate the potentials of Amaranthus hybridus Linn. and Telfairia
occidentalis Hook F. in combating
aspartame– induced oxidative stress in rats.
1.4 OBJECTIVES OF THE STUDY
1.
To determine the effect of leaf methanol extracts of Telfairia occidentalis and Amaranthus hybridus on lipid
peroxidation via malondialdehyde formation in aspartame – treated rats.
2.
To determine the effect of leaf methanol extracts of Telfairia occidentalis and Amaranthus hybridus on the activities of
some antioxidant enzymes namely catalase and superoxide dismutase
3.
To determine the effect of leaf methanol extracts of Telfairia occidentalis and Amaranthus hybridus on the serum lipid
profile of aspartame – treated rats.
4.
To determine the effect of leaf methanol extracts of Telfairia occidentalis and Amaranthus hybridus on the liver enzyme
activities of aspartame – treated rats.
5.
To determine the effect of leaf methanol extracts of Telfairia occidentalis and Amaranthus hybridus on the serum total
protein, albumin and globulins concentrations in aspartame – treated rats.
6.
To determine the effect of leaf methanol extracts of Telfairia occidentalis and Amaranthus hybridus on some kidney
function parameters namely serum urea and creatinine concentrations.
1.5 SCOPE
The
scope of this work covers biochemical assay of the lipid peroxidation marker,
malondialdehyde, the antioxidant enzymes namely; catalase and superoxide
dismutase, lipid profile, liver function and kidney function.
1.6 SIGNIFICANCE OF THE STUDY
This study is significant because it will
give insight into the health effect of excessive consumption of soft and energy
drinks, candies or cookies which are the products in which aspartame is added
as a sweetener to enhance taste. It will also give an insight to the health
benefits /or otherwise of consumption of leafy vegetables particularly Telfairia occidentalis and Amaranthus hybridus used in this study.
Furthermore, it will also contribute to the store of scientific information on
medicinal plants which can be drawn upon by pharmaceutical industries for the
production of new drugs.
1.7 JUSTIFICATION OF THE STUDY
This study is justified by the fact that
there is increasing need to create awareness on the subject of oxidative stress
as the underlying cause of the increasing incidence of chronic debilitating
diseases such as hypertension and stroke, diabetes mellitus, cancers, Alzheimer’s
disease, Parkinson’s disease to mention but a few, which are major causes of
mortality and morbidity in today’s world. There is need to educate people on
ways they can prevent oxidative stress and the accompanying health hazards.
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