ABSTRACT
This study investigated the modulatory effect of ethanol extract of Euphorbia kamerunicus on potassium bromate-induced toxicity in Albino rats. The crude ethanol extract was subjected to phytochemical and, GC-MS analyses and in vitro antioxidant and acute toxicity evaluations. Results obtained following phytochemical studies of the crude extract revealed the presence of alkaloids, saponins, tannins, terpenes, flavonoids, phenols, cardiac glycosides and steroids in various amounts. Alkaloids were the most abundant (21.63±0.15%) while cardiac glycosides were the least (4.60±0.26%). GC-MS analysis revealed the presence of 33 compounds in the crude extract with oleic acid as the most abundant (12.54%) and 2-Propenoic acid the least (0.20%). Other compounds with high abundance in the crude extract were 11-Octadecenoic acid (8.69%), Butyl 9-tetradecenoate (8.63%), n-Decanoic acid (6.98%), 1,3-Dioxolane (9.31%), 6-Octadecenoic acid (7.17%), Methyl stearate (4.27%), 2-Trifluoroacetoxypentadecane (4.54%) and Hexadecanoic acid (3.74%). The crude extract was also fractionated into five fractions. Investigation of the effects of the extract in potassium bromate-induced toxicity was carried out in two phases, first on the crude and then on the fractions. For the crude extract, 30 rats randomly assigned to 6 groups of five rats were used. The rats were treated according to the order: group 1 (normal control with no treatment), group 2 (potassium bromate, 100 mg/kg only), group 3 (200 mg/kg of crude extract + potassium bromate, 100 mg/kg), group 4 (400 mg/kg of crude extract + potassium bromate, 100 mg/kg), group 5 (800 mg/kg of crude extract + potassium bromate, 100 mg/kg) and group 6 (100 mg/kg vitamin C + potassium bromate, 100 mg/kg). Treatment lasted 28 days before animals were sacrificed for haematological and biochemical analyses. In the second phase using the fractions, treatments for groups 1-3 were repeated but groups 4-8 were treated with extract fractions 1-5 for 28 days before sacrifice and analyses of collected samples. Results of in vitro antioxidant activities showed significant nitric oxide and DPPH scavenging activities and mild ferric reducing antioxidant power activity. Acute toxicity value obtained for potassium bromate in rats was 346.41 mg/kg body weight while that of the crude extract was greater than 5000 mg/kg. Results of liver function parameters showed significantly higher AST, ALT and ALP activities in the group administered only potassium bromate when compared with those co-treated with the extract. The crude extract also significantly inhibited anomalies observed in total protein and serum bilirubin values due to potassium bromate intoxication. Higher levels of urea, uric acid and creatinine due to potassium bromate were also significantly lowered in the extract treated groups (p<0.05). Lipid profile values were not significantly altered following treatment with potassium bromate and treatment with the extract (p>0.05), but antioxidant parameters including GSH, GPx, SOD and catalase significantly depreciated with concurrent rise in the bromate only group but ameliorated in the groups treated with the extract (p<0.05). The fall in the values of haematological parameters were also significantly up regulated in the crude-extract-treated groups. Elevated values of cardiac parameters (lactate dehydrogenase, creatine phosphokinase and cardiac troponin) due to bromate intoxication were significantly lowered in groups treated with the crude extract and fractions (p<0.05). Inflammatory markers (interleukin 1b, prostaglandin E2 and tumor necrosis factor) were also lowered significantly (P<0.05). Of all the fractions evaluated, fraction 4 had the higher activities than the other fractions and produced effects similar to that of the crude extract. Cactus plant extract may be of value in the management of potassium bromate-induced systemic toxicity and could be a potential source of control agent for oxidative stress-induced diseases caused by environmental oxidants.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables xii
List of Figures xiv
Abstract xv
CHAPTER ONE
INTRODUCTION 1
1.1
Background of the study 1
1.2
Statement of the problem 4
1.3
Justification for the study 6
1.4
Aim of the
study 6
1.5
Objectives of the study 6
CHAPTER TWO
LITERATURE
REVIEW 8
2.1 Potassium
bromate: an overview 8
2.1.1 Physicochemical
properties 9
2.1.2 Uses
and action 10
2.1.2.1 Toxicity
and Safety 11
2.1.3. Potassium bromated in bread making 16
2.2 Systemic Targets For Potassium Bromate
Induced Toxicity 19
2.2.1 Potassium bromate nephrotoxicity 20
2.2.1.1 Pathophysiologic effect
of KBrO3 in the Liver 23
2.2.2 Potassium bromate induced-oxidative stress 24
2.2.3 Potassium bromate-induced hematological
alterations 26
2.2.4 Potassium bromate-induced cardiac
hypertrophy 27
2.2.5 Pathophysiologic effect of KBrO3
in Other Tissues 27
2.3 Mechanism Action of Potassium Bromate
Toxicity 29
2.4 Effects of Plant-Derived Antioxidants 31
2.4.1 Antioxidants 33
2.4.2 Potassium Bromate and Inflammatory Bowel
Disease 33
2.5 Biology
of Euphorbia kamerunicus 35
2.5.1 Medicinal
benefits 36
CHAPTER THREE
MATERIALS
AND METHODS 37
3.1 Materials (Equipment and Reagents) 37
3.2. Collection and Drying of Plant Materials 37
3.2.1 Preparation of Extract 38
3.3. Qualitative Phytochemical Study of the Extract 38
3.4. Quantitative Phytochemical Tests 40
3.5. In
Vitro Antioxidant Evaluation of the Extract 44
3.6. Determination
of Phytochemical Composition of crude cactus
Extract by Gas Chromatography – Mass Spectrometry (Gc-Ms) 46
3.7. Animals 48
3.8 Acute Toxicity
Studies 48
3.8.1 Acute Toxicity Evaluation of Potassium Bromate 49
3.8.2 Acute Toxicity Evaluation of
Cactus Plant Extract 49
3.9. Experimental Design 50
3.10. Assessment of Haematological Parameters 51
3.11. Assessment of Liver Function Parameters 52
3.12 Assessment of Renal Function Parameters 56
3.13 Lipid Profile Parameters 60
3.14 Assessment of Antioxidant Parameters in
Liver Tissues 63
3.15. Estimation of cardiac parameters (LDH, CPK and cardiac
troponin) 66
3.16. Estimation of inflammatory cytokines (tumor necrotic
factor and
interleukin-1b) 70
3.17 Liver And Kidney Histopathology 74
3.18
Bioassay-Guided Fractionation of Cactus Plant Extract
Using
Chromatographic Techniques 75
3.19
Statistical analysis 80
CHAPTER FOUR
RESULTS 82
4.1 Results of Phytochemical Evaluation 82
4.1.1 Qualitative phytochemical result 82
4.1.2 Quantitative phytochemical result 84
4.2 Result
of Gc-Ms Analysis of Cactus Plant Crude Extract 86
4.3 Antioxidant Activity Assay 90
4.3.1 DPPH free radical scavenging activity 90
4.3.2 Ferric reducing antioxidant potential (FRAP)
assay 92
4.3.3 Nitric oxide (NO) radical scavenging
activity 94
4.4 Results
of Acute Toxicity Evaluation 96
4.4.1 Result of acute toxicity evaluation of
potassium bromate 96
4.4.2 Acute toxicity report of cactus plant
extract 98
4.5 Liver Function Profile 100
4.6 Serum Renal Profile 103
4.7 Serum Lipid Profile Result 107
4.8 Antioxidant Assay 109
4.9 Haematological Profile 112
4.10 Evaluation of Fractions for Cactus 115
4.10.1
Effects of fractions on haematological parameters 115
4.10.2 Effects of fractions on liver function
parameters 119
4.10.3 Effects of fractions on renal function
parameters 122
4.10.4 Effects of fractions on lipid profile
parameters 125
4.10.5 Effects
of fractions on antioxidation enzymes 130
4.10.6 Effects of fractions on cardiac biomarkers 134
4.10.7 Effects of fractions on carcinoma biomarkers 136
CHAPTER
FIVE
DISCUSSION 140
5.1 Phytochemicals 140
5.2 Antioxidant
Potential 142
5.3 Liver
Function Profile 145
5.4 Lipid
Profile 147
5.5 Renal
Function 149
5.6 Hematological
Evaluation 151
5.7 Acute Toxicity 153
5.8 Antioxidants 154
5.9 Cardiac
Markers 156
5.91 Evaluation
of Fractions 157
5.92 Inflammatory
Markers 158
5.93 Conclusion
160
5.94 Recommendations 160
5.95 Suggestion
for Further Study 161
References 162
LIST OF TABLES
Table 4.1a: Qualitative phytochemical components of ethanol extract
of cactus 83
Table 4.1b: Qualitative phytochemical components of ethanol extract
of cactus 85
Table 4.2: GC-MS crude extract 88
Table 4.3a: Acute toxicity evaluation of
potassium bromate phase 1 97
Table
4.3b: Acute toxicity evaluation of potassium bromate phase 2 97
Table 4.4a: Acute toxicity evaluation of ethanol extract of cactus phase 1 99
Table 4.4b: Acute toxicity evaluation of ethanol extract of cactus phase 2 99
Table 4.5: Effects of the ethanol extract of cactus on
some serum biochemical
parameters treated exposed
to potassium
bromate 102
Table 4.6: Effects of the ethanol extracts of cactus on plasma urea, creatinine, uric acid and electrolyte levels
in potassium bromate intoxicated Rats. 105
Table 4.7: Effects of the ethanol extract of cactus on some lipid
profile of
potassium bromate intoxicated Rats. 108
Table 4.8: Effects of ethanol extract of cactus
on protective/antioxidant parameters. 111
Table 4.9: The effects of the extract cactus on some
haematological parameters induced with potassium bromate
intoxicated Rats. 114
Table 4.10: Effect of 5 different fractions and crude extract of cactus
on concentration of some haematological
indices. 117
Table 4.11: Effects of five different
fractions and crude extract of cactus
on concentration of selected
liver function parameters. 120
Table 4.12: Effects of five different fractions and crude extract of
cactus on selected renal function parameters. 125
Table 4.13: Effect of 5 different fractions
and crude extract of cactus
on
selected lipid parameters. 128
Table 4.14: Effects of five different fractions and crude extract of cactus
on redox status of
potassium bromate intoxicated Rats 132
Table 4.15: Effects of five different
fractions and crude extract of cactus
on some cardiac
biomarkers of potassium bromate intoxicated Rats.136
Table 4. 16: Effects
of five different fractions and crude extract of cactus
plant
on the concentration levels some carcinoma biomarkers exposed
to
potassium bromate. 139
LIST OF FIGURES
Figure 2.1
Structure of the Potassium Bromate Molecule 10
Figure 4.1: GC-MS chromatogram showing 33 compounds
in cactus
plant crude extract. 87
Figure
4.2a: DPPH free radical scavenging antioxidant activity. 91
Figure
4.2b: Ferric reducing antioxidant power (FRAP) assay. 93
Figure
4.2c: Nitric oxide radical scavenging activity. 95
CHAPTER
ONE
INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
Given the expanding
horizons of food habits, the exposure of mankind to xenobiotics necessitates novel
and undefined biological interactions, including the damaging effects in the
living system, making it one of the predominant health concerns in the public
domain in the modern times. Potassium bromate (KBr03) is a food
additive that has been extensively used in food, cosmetic, and pharmaceutical
industries, since its discovery in 1900. Potassium bromate is an oxidising
agent and one of the best and cheapest dough improvers in the baking industry. (Altoom
et al., 2018).
It plays a major role in the bread-making
industry. Potassium bromate (PB) has significant effect on food biomolecules,
such as starch and protein, as it affects the extent of gelatinisation,
viscosity, swelling characteristics as well as gluten proteins; it removes
sulphhydyl group and leads to the formation of disulphide linkages and thus
improves bread properties. However, there are many reports on its deleterious
impact on human health. It is a potential human carcinogen according to International
Agency for Resaerch on Cancer (IARC). Ajarem et al., 2016; Ahmad et al.,
2015). Due to this, countries across the world have either partially or
completely prohibited its usage. Numerous techniques have evolved to determine
the concentration of potassium bromate in bread. It has also been found to be
used in the production of beer, cheese, and fish paste products at the
industrial level (Altoom et al.,
2018; Ajarem et al., 2016; Ahmad et al., 2015).
Reactive oxygen species
(ROS) and free radicals have been implicated in mediating KBrO3-induced
toxicity. These radicals induce tissue damage by reacting with macromolecules
like proteins, nucleic acids, and lipids, leading to tissue injury (Ahmad et al., 2012 and 2013).
It degrades vitamins A, B1, B2 and niacin which are the main vitamins
available in bread and has been classified by the International Agency for
Research on Cancer (IARC) as a possible human carcinogen based on sufficient
evidence that KBrO3 is carcinogenic and mutagenic in experimental animals
(Fawell and Walker, 2006). According to the United States Department of
Agriculture (USDA), it improves dough processing properties, internal crumb
quality and low volume in concentration from a few to 75 ppm, the highest
concentration permitted by law. In early 1990’s, the World Health Organization
(WHO) discovered that potassium bromate if consumed has the capacity to cause
such diseases as cancer, kidney failure and several other related diseases. (Fawell
and Walker, 2006).
Moreover, traces of KBR03
have also been found in various packaged drinking and municipality-treated tap
water which comes after ozonization of water during its filtration process.
Hence, its wide usage put humans at risk as KBRO3 induces mild to
severe toxic damages depending on the dose and duration of exposure. It is
documented that it causes multiple organ toxicities upon internalisation in a
living system; however, the kidney, liver, and brain are the major target
organs (Ben Saad et al. 2015; Ahmad
and Mahmood, 2012). It has also been reported that PB elicits free radical
generation during its biotransformation leading to elevation in oxidative
stress, extensive tissue damage, macromolecular disruption, and, even, cancer which
is contingent on the dose, duration, and concurrent circumstances in the living
systems (Ahmad et al., 2015; Ajarem et al., 2016). The increasing global
trend of the bakery-based western fast food (pizza, burger, cake, etc.) and
many of the commercial soft drinks that contain KBRO3 as an additive
and taste enhancer expose the next generation at high risk of KBR03
induced toxicities. The market share of other forms of this compound as
cosmetics, chemical preservatives, and drugs, is also expanding with time.
Despite the commercial
value of potassium bromate (KBrO3), in food and cosmetics industries
and being a drinking water disinfection by-product, it has been and remains of
paramount concern for human health (Gosh et
al., 2017). A study giving a new insight into oxidative
stress- related in vivo mutagenicity and genotoxicity exerted
by KBrO3 was reported (Tsuchiya et al.,
2018; Obaidi et al., 2018).
Similarly, it was reported that genotoxic effects of KBrO3 were
associated with DNA and chromosomal damages, mutations, base modifications,
chromosomal aberrations, and altering gene expression, leading to cancer
(Spassova et al., 2013; Chauhan and Jain, 2016).
Elmahdy et al. (2015) opined that exposure to
KBrO3 elicited lesion to liver tissues and also heightened
serum enzyme levels. In another study, KBrO3 was demosntrated
to induce cytotoxicity in testicular cells which were inhibited by antioxidants
(Nwonuma et
al., 2016). These outcomes were found to be in tandem with those
obtained by Elsheikh et al. (2016).
Cytotoxic effects of KBrO3 on kidney cells of Fischer 344 rats
were also reported (Dodd et al.,
2013). Stuti and D’souza (2013) reported the toxic effects of KBrO3 on
biochemical, haematological, and histological parameters of Swiss albino mice.
These findings were corroborated by another study which demonstrated that
exposure to KBrO3 causes cell lysis in human erythrocytes (Ahmad
et al., 2014).
Cactus
(Opuntia ssp.) is a medicinal plant. There are about 200 recognised species of
Opuntia. These plants contain a wide variety of trace elements, sugars and
other bioactive compounds, such as betalains, carotenoids, ascorbic acid,
flavonoids and other phenolic compounds. Cactus
plants are now considered as a rich source of nutritional compounds with
health-promoting activities, including antioxidant, neuroprotective,
cardioprotective, anti-inflammatory, anti-diabetic, anti-clastogenic and
anti-genotoxic actions. On the basis of the properties
of the active compounds contained in Cactus plant, it is thought that the plant may have a potential protective role against
oxidative stress, genotoxicity, and cytotoxicity in animals.
They also protect
erythrocyte membranes and acute gastric lesions, and improve platelet function
and used in cancer chemoprevention (González-Ponce et al., 2016; Han et al.,
2017).
1.2 STATEMENT
OF THE PROBLEM
According to Oyekunlea et al., (2014), the American Food and
Drug Administration (FDA) allows the use of KBRO3 up to a maximum
level of 50 mg/kg of flour mass in bread, while, Japan only permits the
inclusion only up to 10 mg/kg of flour. (Oyekunlea et al., 2014). In California,
some state of the USA, a warning label is required when bromated flour is used,
while some insist that it is inappropriate to use KBRO3 in any
product or production method, which can be formulated with residues below the
level of 20 ppb (i.e. 0.020 mg/kg) in the finished product (ABA/AIBI (2008).
The Food and Agriculture Organization (FAO)/World Health Organization (WHO) joint
committee’s initial recommendation of acceptable level of 0 - 60 mg KBrO3/kg
flour was later withdrawn because of
long term toxicity and carcinogenicity studies (in vitro and in vivo), which had revealled the development of renal
cell tumors in hamsters.
An investigation into the
health dangers of potassium bromate in especially bread reveals the harmful
effects potassium bromate has on consumers, leading to its ban in 1993, by the
Federal Ministry of Health. Potassium bromate (KBrO3) is an
oxidizing agent that has been used as a food additive, mainly in the
bread-making process. It has been demonstrated that potassium bromate induces
renal cell tumors, mesotheliomas of the peritoneum, and follicular cell tumors
of the thyroid. In addition, experiments aimed at elucidating the mode of
carcinogenic action have revealled that it is a complete carcinogen, possessing
both initiating and enhancing activities for rat renal tumorigenesis. Numerous
other studies have revealled the potential of potassium bromate to lead to cancer
in experimental animals and in humans. In Nigeria, bromate as bread improver
has been banned (Ekop et al., 2008). However,
some bread makers/bakeries have continued to include potassium bromate in their
bread making process, simply because it enhances their profit mergins.
1.3 JUSTIFICATION
FOR THE STUDY
Plant derived products
have been used for medicinal purposes for centuries and presently, it is
estimated that about 80% of the world population relies on botanical
preparations as medicines to meet their health needs. This may be attributable
to the down turn in the economy, as herbal medicine is perceived to be a
cheaper means of treatment (Shri, 2003), hence this
study on the use of Euphorbia kamerunicus extract as a modulator of
the adverse effects of KBRO3 in
intoxicated rats.
1.4 AIM OF
THE STUDY
The aim of this study was
to investigate the effect of Euphorbia
kamerunicus cactus ethanol
extract and fractions on potassium bromate- induced toxicity in albino rats.
1.10
OBJECTIVES
OF THE STUDY
The
objectives of the study were to:
i.
Determine the qualitative
and quantitative phytochemical composition of cactus plant extract by manual techniques and by GC-MS.
ii.
Evaluate the acute
toxicity of cactus crude extract and
potassium bromate.
iii.
Evaluate the effects of ethanol
extract cactus and fractions on the
haematological indices in potassium bromate toxicity in rats.
iv.
Evaluate the effects of
ethanol extract of cactus and fractions on serum biochemical parameters
including liver function, renal function, lipid profile and redox status in
potassium bromate indused toxicity in rats.
v.
Evaluate the effects of
cactus plant crude extract and fractions on the cardiac troponin, lactate
dehydrogenase and creatine phosphokinase levels in potassium bromate indused
toxicity in rats.
vi.
Evaluate the effects of cactus crude ethanol extract and
fractions on inflammatory cytokines
including prostaglandin E2, interleukin-1b and tumor necrosis factor in
potassium bromate- intoxicated rats.
vii.
Determine the most active
fraction of cactus crude extract.
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