ABSTRACT
Liver and kidney damages are of growing concern to today’s modern society. The increasing incidence of exposure to toxic agents has contributed to liver and renal diseases. There is therefore, need for hepatoprotective and renal agents. This study was aimed at investigating the hepatoprotective and renal effects of methanol extract of Napoleonae imperialis in albino rats. The phytochemical analysis and acute toxicity studies (LD50) of the extract were also evaluated using standard methods. Forty eight (48) male albino rats of mean weight 130 g were used for this study. Twenty four (24) male albino rats were used for the hepatic studies, while the other twenty four rats were used for the renal studies. The animals for the study were grouped into eight (8) of six (6) rats each. Groups A and B were the control groups, while groups C and D were the test groups. Group A received feed and water only and Group B was induced with carbon tetrachloride (CCl4) without treatment. Test groups (C and D) were orally given 250 mg and 500 mg/kg body weight of Napoleonae imperialis leaves extract respectively for 14 days. All the rats used in this study (hepatic study) were initially subjected to hepatocyte damage using 2.0 ml/kg of carbon tetrachloride (CCl4) except the normal control group. For the renal studies, group E represented the positive control that was induced with methotrexate without treatment. Groups (F and G) were the test groups that received orally 250 mg and 500 mg/kg body weight of the plant extract respectively, while Group H was the group that only received orally 500 mg/kg b.wt of the plant extract. All the rats used in this study (renal study) were initially subjected to renal damage using 0.5 ml/kg of methotrexate. The rats were sacrificed after 14 days and the blood samples were collected for biochemical analysis. The liver and renal function tests were performed, in addition to their histopathological evaluation. Phytochemical analysis showed the presence of carbohydrates, reducing sugar, glycosides, acidic compounds, tannins, phenols, flavonoids, steroids, terpenoids, and alkaloids. The acute toxicity study (LD50) showed no adverse effect in their general behaviour, and mortality at the dose level given (100- 5000 mg/kg b.wt). The result obtained showed a significant decrease (p< 0,05) of the liver parameters (AST, ALT, ALP, albumin, total protein and total cholesterol) in the test groups treated with 250 mg and 500 mg/kg body weight of the plant extract, unlike the positive control where the liver parameters were still high which could be as a result of hepatocellular damage by carbon tetrachloride (CCl4) without treatment. Also the comparison of the mean difference of the test groups and the control groups (normal and positive control) revealed that the methanol extract of Napoleonae imperialis caused a significant (p< 0.05) decrease in urea, creatinine, N+, K+, and Cl- There was also a significance (p< 0.05) decrease in the hepatic and renal parameters in comparison of the mean difference of the control groups (normal and positive control) and the group that received the extract only (500 mg/kg b.wt). The results of this study indicate that the methanol extract of Napoleonae imperialis may have exerted hepatoprotective and renal functioning effect in albino rats, and may also be used pharmacologically in the management of organ toxicity.
TABLE OF CONTENTS
Title
page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of tables viii
List of figures ix
List of plates x
List of symbols and abbreviations xi
Abstract xii
CHAPTER 1 INTRODUCTION
1.1 Background of
the Study 1
1.2 Justification 2
1.3
Scope of the Study 2
1.4
Aim of the Study 2
1.5
Specific Objectives 3
1.6
Significance of Study 3
1.7 Research
Questions 3
CHAPTER 2 LITERATURE
REVIEW
2.1 BACKGROUND – Napoleonae imperialis 5
2.1.1 Distribution 6
2.1.2 Medicinal
use of Napoleonae imperialis 6
2.1.3
Taxonomy 7
2.1.4 Common name 7
2.2 Hepatotoxicity 7
2.2.1 Hepatotoxic agents 9
2.2.2 Hepatoprotective agents 10
2.2.3 Common medicinal plants having
hepatoprotective activity. 11
2.3 Mechanism of
Action of Haloalkane (chloroform hexane) in Hepatocellular Damage 12
2.4 Major
Functions of the Liver 13
2.5 Role of Free
Radicals in Liver Toxicity 13
2.6 Liver Function
Test 14
2.7 Markers of
Liver Functions 15
2.7.1 Alanine aminotransferases (ALT) 15
2.7.2 Aspartate aminotransferases (AST) 16
2.7.3 Total protein 16
2.7.4 Albumin 16
2.7.5 Gamma
glutamic transpeptidase (GGT) 17
2.7.6 Total cholesterol 17
2.8 Functions of Kidney 17
2.8.1 Epidemiology 18
2.8.2 Pathophysiology of renal failure in liver disease 19
2.8.3
Causes of acute renal failure in liver disease 21
2.8.3.1
Pre- renal 21
2.8.3.2
Intrinsic renal 22
2.8.3.3 Acute tubular necrosis
(ATN) 22
2.8.4 Management of acute renal failure (ARF) in liver disease 24
2.8.5 Prevention
of renal failure in advanced liver disease 26
2.8.5.1
Acute renal failure post liver resection and liver transplantation 27
2.9
Nephrotoxic Agents 28
2.9.1
Nephropathies 29
2.10 Electrolytes
and Renal Function 30
CHAPTER 3 MATERIALS
AND METHODS
3.1
Equipment 32
3.2 Chemicals 32
3.3 Plant Material 32
3.4 Extraction 33
3.5 Animals 33
3.6 Induction of
Hepatoxicity and Nephrotoxicity 33
3.7 Experimental Design 33
3.8 Preliminary Phytochemical Screening 34
3.9
Acute Toxicity Studies 34
3.10 Evaluation of
the Various Parameters Studied 35
3.10.1 Assay for
alanine aminotransferase (ALT) 35
3.10.2 Assay for aspartate transaminase
(AST) 36
3.10.3 Determination
of alkaline phosphatase (ALP) 36
3.10.4 Determination of serum total
protein 37
3.10.5 Determination of albumin 38
3.10.6
Determination of serum creatinine concentration 38
3.10.7
Determination of serum urea concentration 39
3.10.8 Determination of total cholesterol 40
3.11
Tests for Electrolyte Activity 41
3.11.1
Estimation of sodium ion (Na⁺) and potassium
ion (k⁺) 41
3.11.2
Estimation of serum chloride concentration 42
3.12 Histopathological Examination 43
3.13
Statistical Analysis 43
CHAPTER 4 RESULTS
AND DISCUSSION
4.1 Acute Toxicity and Lethality Test 45
4.2 Effects of Methanol Extract of Napoleonae imperialis on the Liver
and
Kidney Parameters 47
4.3 Histological Effects of Methanol
Extract of Napoleonae imperialis on
the Liver 69
4.4 Histological
Effects of Methanol Extract of Napoleonae
imperialis on the Kidney 74
4.5 Discussion 79
CHAPTER 5 CONCLUSION
AND RECOMMENDATIONS
5.1 Conclusion 85
5.2
Recommendations 85
References 86
Appendices 97
LIST OF TABLES
2.0 Common
medicinal plants having hepatoprotective activity 11
4.1 Qualitative composition of powdered Napoleonae imperialis leaf extract 44
4.2 Quantitative composition of powdered Napoleonae imperialis leaf extract 45
4.3 LD50 of the methanol extract of Napoleonae imperialis in albino rats 46
LIST OF FIGURES
2.0 Diagram of Napoleonae imperialis plant 5
4.1 Effect of methanol extract of Napoleonae imperialis on AST 47
4.2 Effect of methanol extract of Napoleonae imperialis on ALT 48
4.3 Effect of methanol extract of Napoleonae imperialis on ALP 49
4.4 Effect of methanol extract of Napoleonae imperialis on Total protein 50
4.5 Effect of methanol extract of Napoleonae imperialis on Albumin 51
4.6 effect of methanol extract of Napoleonae imperialis on Total
cholesterol 52
4.7 Effect of methanol extract of Napoleonae imperialis on AST (mean
comparison between
the control groups and the group that received the extract only) 53
4.8 Effect of methanol extract of Napoleonae imperialis on ALT (mean
comparison
between the control groups and the
group that received the extract only) 54
4.9 Effect of methanol extract of Napoleonae imperialis on ALP (mean
comparison between
the control groups and the group that received the extract only) 55
4.10
Effect of methanol extract of Napoleonae
imperialis on Total protein (mean
comparison between the control
groups and the group that received the extract) 56
4.11
Effect of methanol extract of Napoleonae
imperialis on Albumin (mean
comparison
between the control groups and the group that received the extract) 57
4.12
Effect of methanol extract of Napoleonae
imperialis on Total cholesterol (mean
comparison between the control
groups and the group that received the extract 58
4.13
Effect of methanol extract of Napoleonae
imperialis on Urea 59
4.14 Effect of methanol extract of Napoleonae imperialis on Sodium ion (Na+) 60
4.15
Effect of methanol extract of Napoleonae
imperialis on Chloride ion (Cl-) 61
4.16
Effect of methanol extract of Napoleonae
imperialis on Creatinine 62
4.17
Effect of methanol extract of Napoleonae
imperialis on Potassium ion (k+) 63
4.18
Effect of methanol extract of Napoleonae
imperialis on Urea (mean
comparison between the control
groups and the group that received the extract) 64
4.19
Effect of methanol extract of Napoleonae
imperialis on Sodium ion (mean
comparison
between the control groups and the group that received the extract) 65
4.20
Effect of methanol extract of Napoleonae
imperialis on Chloride ion (mean
comparison between the control
groups and the group that received the extract 66
4.21
Effect of methanol extract of Napoleonae
imperialis on Creatinine (mean
comparison
between the control groups and the group that received the extract) 67
4.22
Effect of methanol extract of Napoleonae
imperialis on Potassium ion (mean
comparison
between the control groups and the group that received the extract 68
LIST OF PLATES
4.1 A cross
section of the liver of normal control group H&E x400 69
4.2 A cross
section of the liver of positive control (CCl4) intoxicated
without
treatment) H&E x400 70
4.3 A cross
section of the liver of CCl4 intoxicated rats
treated
with 250 mg/kg body weight of Napoleonae
imperialis leaf extract H&E 71
4.4 A cross
section of the liver of CCl4 intoxicated rats
treated
with 500 mg/kg body weight of N.
imperialis leaf extract H&E x400 72
4.5 A cross
section of the liver treated with 500 mg/kg body
weight
of N. imperialis leaf extract only
H&E x400 73
4.6 A cross
section of the kidney of normal control group H&E x400 74
4.7 A cross
section of the kidney of positive control (methotrexate
intoxicated
rats without treatment) H&E x400 75
4.8 A cross
section of the kidney of methotrexate intoxicated
rats
treated with 250 mg/kg body weight of N.
imperialis H&E x 400 76
4.9 A cross
section of the kidney of methotrexate intoxicated rats
treated
with 500 mg/kg of body weight of N.
imperialis leaf extract H&E x400 77
4.10 A cross
section of the kidney treated with 500 mg/kg of
body
weight of N. imperialis leaf extract
only H&E x400 78
LIST OF SYMBOLS & ABBREVIATIONS
Abbreviation
Notation
- mg Milligram
- g
Gram
- kg
Kilogram
- mg/dl
Milligram per decilitre
- dl Decilitre
- %
Percentage
- ALP
Alkaline phosphatase
- AST Aspartate transaminase
- ALT
Alanine aminotransferase
- GGT
Gamma glutamic transpeptidase
- CCl4
Carbon tetrachloride
- MTX Methotrexate
- TAG
Triacylglycerol
T. chol
Total cholesterol
- Cl-
Chloride ion
- Na+
Sodium ion
- K+
Potassium ion
- TP Total
protein
- ALB
Albumin
- LD50 Acute toxicity and
lethality dose
CHAPTER 1 INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
Liver and kidney malfunction frequently existing
collectively, both as part of multiorgan dysfunction in an unfavourably sick
patient, or due to damage of each organ individually. Three principal medical
conditions can be recognized in which liver and kidney malfunction coincide;
infections concurrently relating the liver and the kidney, or a basic liver
disorder with elementary kidney malfunction, or vice versa (Moreau et al., 2002). Attendant liver and
kidney abnormality may exhibit mutual pathogenetic mechanisms. kidney
malfunction in this scenario often grows gradually, with the exclusion of
definite disease such as leptospirosis, some viral hemorrhagic fevers and
toxin-mediated damages such as paracetamol injury, which trigger severe failure
of both organs (Eckardt, 2000). kidney dysfunction relative to hepatic
malfunction is usually efficient in nature and arises due to the absence of
important modifications in kidney histology (pre-renal). However, inherent
kidney dysfunction may lead to severe or prolonged hepatic disease (inherent
kidney failure) (Moreau et al., 2002).
Preventive uropathy could result to postrenal severe kidney dysfunction which
often manifest in chronic hepatic disease (papillary necrosis in alcoholic
liver disease, haemorrhage in the urinary route as a result of acute
coagulopathy) (Ifediora and Benz, 2003). Hepatorenal disease is a major type of
practical kidney malfunction that frequently result in progressive hepatic
pathology, kidney disorder or threshold hypertension (Guevara and Gines, 2005).
Liver and kidney damage
could be triggered by several toxic agents like certain antibiotics,
chemotherapeutic agents, carbon tetrachloride (CCl4), thioacetamide
(TAA), methotrexate (MTX), other haloalkanes, excessive alcohol consumption and
microbes is well studied.
Consequently,
in this study we monitored the hepatoprotective potentials and renal effects of
methanol extract of Napoleonae imperialis
in albino rats with a view to understanding at least in part, the mode by which
methanol extract of Napoleonae imperialis
mediate or exert their hepatoprotective effects and renal functions in albino
rats.
1.2 JUSTIFICATION
OF THE STUDY
The
justification of this study is due to the fact that most people often use
extracts of Napoleonae imperialis to
cure wounds and hypertension. The hypotensive effect could be attributed to the
activation of muscarinic receptors of the parasympathetic nerve by the
compounds or their actions as an antagonist of adrenergic receptor, but it may
act as Ca2+ ion channel block. For this reason, it was also
suspected that the plant may possess hepatoprotective and renal effects in
albino rats. The study is therefore set to accept or reject this suspicion.
1.3
SCOPE OF THE STUDY
This work covers the
assessment of hepatoprotective effects and renal function of oral
administration of methanol extract of Napoleonae
imperialis in albino rats.
1.4
AIM OF THE STUDY
To evaluate the
hepatoprotective effects and renal function of methanol extract of Napoleonae imperialis in albino rats.
1.5
SPECIFIC OBJECTIVES
Specifically, the study
sort
(i)
To determine the qualitative and quantitative phytochemical properties of
powdered leaves extract of Napoleonae
imperialis in albino rats.
(ii)
To determine the LD50 of the methanol extract of Napoleonae imperialis in albino rats.
(iii)
To evaluate the effect of methanol extract of Napoleonae imperialis on the liver markers (ALT, AST, ALP), total
protein, and albumin in albino rats
(iv)
To evaluate the effect of methanol extract of Napoleonae imperialis on the renal function markers (urea,
creatinine, Na+, and K+) in albino rats.
1.6 SIGNIFICANCE
OF THE STUDY
This
research will be significant in the following ways:
(i)
In evaluating the outcome of methanol extract of Napoleonae imperialis on hepatoprotective potentials and renal
function of albino rats.
(ii)
The result obtained could prove valuable for further research on the leaves
extract and the recommendation of the plant as a choice of traditional medicine
in the treatment of hepatic and renal damage.
1.7 RESEARCH
QUESTIONS
(i)
Does methanol extract of Napoleonae
imperialis boost the level or activity of hepatoprotective potentials in
albino rats?
(ii)
Does methanol extract of Napoleonae imperialis enhance the renal
function of albino rats?
(iii)
At what concentration does the methanol extract of Napoleonae imperialis confer hepatoprotective potentials and renal
functioning effect in albino rats?
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