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PHYTOCHEMICAL, ANTIOXIDANT AND TOXICOLOGICAL PROPERTIES OF METHANOL EXTRACT OF PHYLLANTHUS AMARUS

Bio Chemistry Project Topics & Materials

ABSTRACT
The use of medicinal plants for the treatment of many diseases is associated with folk medicine from different parts of the World. However, information on the toxicology of these plants part used in Nigeria folk medicine is rare. Thus, this work is aimed at revealing a range of phytochemicals in the Phyllanthus amarus plant, the antioxidant constituents and its toxicologic effects on some important biochemical parameters in male albino rats. The potent bioactive agents in the leaves of Phyllanthus amarus plant were extracted and the antioxidant and toxicological potentials for in vitro analyses of the crude plant extract were evaluated using in vitro methods and male white albino rats as the model. The results showed that methanol extract scavenged 1,1- diphenyl-2- picryhydrazyl radical (DPPH) in a concentration – dependent manner with a correlation coefficient (R2) of 0.989, indicating antioxidant activity with effective concentration that inhibits fifty percent of the radical (EC50) of 6.93µg/ml compared to ascorbic acid standard with EC50 of 4.69µg/ml. Superoxide radical scavenging activity was concentration dependent with an EC50 of 5.01µg/ml compared with ascorbic acid standard with EC50 of 4.49µg/ml. The crude extract also showed hydroxyl radical scavenging activity with an EC50 of 6.47µg/ml compared to α – tocopherol standard with EC50 of 5.73µg/ml. The methanol extract, also scavenged nitric oxide radical in a concentration – dependent manner with 600µg/ml being more potent than 600µg/ml of α – tocopherol standard. Comparison of the anti-radical power (ARP) of DPPH (0.144), superoxide radical (0.199) and hydroxyl radical (0.175) of the extract revealed that the ARP of the extract against superoxide radical was most efficacious. The antioxidant vitamin contents of the extract showed that vitamin C was significantly higher (p ˂ 0.05), 1.65mg/100g when compared to vitamin A (1.52mg/100g) and vitamin E (0.89mg/100. Acute toxicity test was conducted using mice and there was no death recorded in the mean lethal dose (LD50) investigation. The 100, 200 and 400 mg/kgbw fed to rats showed significantly higher activity of catalase (p ˂ 0.05) at week two and week four. The aspartate aminotransferase (AST) showed non- significantly lower activity (p > 0.05) in group 3 of week one and four, while group 3 of week two was significantly higher (p ˂ 0.05) in week four. The alanine aminotransferase (ALT) indicated a relatively lower activity of ALT from week one to three while there was relative elevation of ALT activity in the test group of week four. The serum alkaline phosphatase (ALP) was significantly lower (p > 0.05) in the test group when compared to the control group 1 in week one. At week two and three, there were higher activities of ALP in all groups, though non- significant while in week four, there was a non- significantly lower activity of the enzyme in all groups. The serum urea concentration showed a significantly higher (p ˂ 0.05) level in all groups except group four in week one. In week two and three, there was a significantly higher level (p ˂ 0.05) while week four exhibited a non-significant increase in serum urea concentration in all groups. The creatinine concentration indicated a significantly higher level (p ˂ 0.05) in groups 2, 3 and 4 in week one. At week two, there was a significantly lower level (p > 0.05) in group two and four. In week three, there was a significantly higher concentration (p ˂ 0.05) in group two and four, while in week four; there was a non- significant difference in the concentration of serum creatinine in all groups. The Packed cell volume (PCV) and haemoglobin count were significantly higher (p ˂ 0.05) in all groups in week one. In week two, there was no significant increase (p > 0.05) in group three. In week three, there was a significantly higher level of PCV and Hb respectively (p ˂ 0.05). Week four indicated a non- significant decrease in all groups. White blood cell count showed a significantly higher level in group 3 and 4 (p ˂ 0.05) except group two in week one. In week two and three, there was an increase in group three while others showed no significant difference. In week four, there was a non – significant decrease in all groups. Histological analysis showed some level of toxicity in 100, 200 and 400mg/kgbw at beyond 14 days of administration. These results seem to suggest rich phytochemical constituents, moderate antioxidant activity, relatively safe level at acute phase (within 14 days) and some level of toxicity in enzyme activity at the chronic phase (after the 14 days of administration).

TABLE OF CONTENTS
Page
Title Page i
Certification ii
Dedication iii
Acknowledgement iv
Abstract v
Table of Contents vi
List of Figures xii
List of Tables xiii
List of Plates xiv
List of Abbreviations xv

CHAPTER ONE: INTRODUCTION
1.1 Profile of Phyllanthus amarus 1
1.2 Phytochemistry 2
1.2.1 Alkaloids 3
1.2.2 Flavonoids 3
1.2.3 Saponins 3
1.2.4 Glycosides 4
1.2.5 Tannins 4
1.2.6 Essential Oils 5
1.2.7 Total Phenolics 5
1.3 Reactive Oxygen Species 6
1.4 Acute Toxicity 8
1.5 Antioxidants 8
1.5.1 Antioxidant Vitamins 9
1.5.2 Catalase 10
1.6 1, 1- Diphenyl-2- picryhydrazyl radical (DPPH) assay 10
1.7 Liver Function Tests 11
1.8 Kidney Function Tests 12
1.8.1 Serum Electrolytes 12
1.8.1.1 Sodium 12
1.8.1.2 Potassium 13
1.8.1.3 Chloride 13
1.9.0 Haematology 13
1.10 Histopathology 14
1.11 Aim and Objective 14
1.11.1 Aim 14
1.11.2 Objectives 14

CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials 16
2.1.1 Plant Materials (Phyllanthus amarus) 16
2.1.2 Animals 16
2.1.3 Chemicals/ Reagents 16
2.1.4 Instruments/ Equipment 16
2.2. Methods 17
2.2.1 Experimental Design 17
2.2.1.1 Extraction of Phyllanthus amarus 17
2.2.1.2 Percentage Yield of Phyllanthus amarus 17
2.2.1.3 Acute Toxicity Tests: Lethal Median Dose (LD50) Determination 18
2.2.1.4 Chronic Toxicity Tests 18
2.2.3 Phytochemical Analysis 19
2.2.3.1 Protein (Millon’s Test) 19
2.2.3.2 Alkaloids (General Test); Wagner’s Test and Mayer’s Test 19
2.2.3.3 Carbohydrate (Molisch’s Test) 19
2.2.3.4 Flavonoids (Ammonium Test Method) 19
2.2.3.5 Saponins 19
2.2.3.6 Glycosides (Fehling’s Test) 20
2.2.3.7 Reducing sugar 20
2.2.3.8 Tannins (Ferric Chloride) 20
2.2.3.9 Acid Test 20
2.2.3.10 Test for Oil 20
2.2.4 Quantitative Phytochemical Analysis 20
2.2.4.1 Determination of Total Phenolic Contents 21
2.2.4.2 Determination of Tannin Contents 21
2.2.4.3 Determination of Flavonoids and Flavonols 21
2.2.5 Antioxidant Vitamins 22
2.2.5.1 Vitamin A 22
2.2.5.2 Vitamin E 22
2.2.5.3 Vitamin C 23
2.2.6 In vitro Antioxidant assays 24
2.2.6.1 Qualitative DPPH Radical Scavenging Assay 24
2.2.6.2 Quantitative DPPH Radical Scavenging Assay 24
2.2.6.3 Hydroxyl Radical (OH-) Radical Scavenging Assay 25
2.2.6.4 Superoxide Scavenging Assay 26
2.2.6.5 In vitro Nitric Acid Radical Scavenging Assay 26
2.2.6.6 Catalase 27
2.2.7 Liver Function Tests 28
2.2.7.1 Assay of Alanine Aminotransferase (ALT) Activity 28
2.2.7.2 Assay of Aspartate Aminotransferase (AST) Activity 29
2.2.7.3 Assay of Alkaline Phosphatase (ALP) Activity 30
2.2.8 Kidney Function Tests 30
2.2.8.1 Determination of Urea Concentration 30
2.2.8.2 Determination of Creatinine Concentration 31
2.2.9 Serum Electrolytes 32
2.2.9.1 Determination of Sodium ion Concentration 32
2.2.9.2 Determination of Potassium ion Concentration 33
2.2.9.3 Determination of Chloride ion Concentration 34
2.2.10 Haematology 35
2.2.10.1 Packed Cell Volume (PCV) 35
2.2.10.2 Haemoglobin Estimation 35
2.2.10.3 Total White Blood Cell (WBC) Count 36
2.2.11 Histological Examination 36
2.2.12 Statistical Analysis 38

CHAPTER THREE: RESULTS
3.1 Percentage Yield of Phyllanthus amarus 38
3.2 Phytochemical Composition of Phyllanthus amarus 38
3.3 Effect of Methanol Extract of Phyllanthus amarus (MEPA)
on DPPH Radical Scavenging Activity 39
3.4 Effect of Methanol Extract of Phyllanthus amarus (MEPA) on Superoxide
Radical Scavenging Activity 41

3.5 Effect of Methanol Extract of Phyllanthus amarus (MEPA) on Hydroxyl
Radical Scavenging Activity 42
3.6 Effect of Methanol Extract of Phyllanthus amarus (MEPA) on Nitric Oxide
Radical Scavenging Activity 43
3.7 Comparison of the Anti- Radical Power of the Extract against DPPH,
Superoxide Radical and Hydroxyl Radical Scavenging Activity 44
3.8 Antioxidant Vitamin Contents of MEPA 45
3.9 Acute Toxicity 46
3.10 Effect of MEPA on In Vivo Catalase Activity 47
3.11 Effect of MEPA on Serum Alanine Aminotransferase (ALT) Activity 48
3.12 Effect of MEPA on Serum Aspartate Aminotransferase (AST) Activity 49
3.13 Effect of MEPA on Serum Alkaline Phosphatase (ALP) Activity 50
3.14 Effect of MEPA on Serum Urea 51
3.15 Effect of MEPA on Serum Creatinine Level 52
3.16 Effect of MEPA on Serum Sodium Level 53
3.17 Effect of MEPA on Serum Potassium Level 54
3.18 Effect of MEPA on Serum Chloride 55
3.19 Effect of MEPA on Haemoglobin Count 56
3.20 Effect of MEPA on White Blood Cell Count 57
3.21 Effect of MEPA on Catalase Activity 58
3.22 Histopathological Examination on the control Group 1; Liver 59
3.23 Histopathological Examination on the control Group 1; Kidney 60
3.24 Histopathological Examination on the 100mg/Kg bw; Liver (Group Two) 61
3.25 Histopathological Examination on the 100mg/Kg bw; Kidney (Group Two) 62
3.26 Histopathological Examination on the 200mg/Kg bw; Liver (Group Three) 63
3.27 Histopathological Examination on the 200mg/Kg bw; Kidney (Group Three) 64
3.28 Histopathological Examination on the 400mg/Kg bw; Liver (Group Four) 65
3.29 Histopathological Examination on the 400mg/Kg bw; Kidney (Group Four) 66

CHAPTER FOUR: DISCUSSION
4.1 Discussion 69
4.2 Conclusion 73
4.3 Suggestions for Further Studies 73
REFERENCES 74
APPENDICES

LIST OF FIGURES
Fig. 1 Phyllanthus amarus 2
Fig.2 Structure of some secondary metabolites/ bioactive agents. 6
Fig .3 Structures of 1,1-diphenyl-2-picrylhydrazyl radical and
1,1-diphenyl-2-picrylhydrazine 10
Fig .4 The nitric oxide radical scavenging activity of MEPA in bar charts 48
Fig. 5 Bar chart representing different anti radical power (ARP) of the extract against the
different free radicals used. 50
Fig. 6 Bar chart of different concentrations of antioxidant vitamins as
determined by the in vitro estimation of the vitamins 52
Fig. 7 Bar chart showing the in vivo catalase activity of MEPA 56
Fig. 8 The results of the effect of MEPA on serum Alanine
Aminotransferase of albino rats 58
Fig .9 The results of the effect of MEPA on serum Aspartate
Aminotransferase of albino rats 60
Fig. 10 The effect of MEPA on serum Alkaline Phosphatase of albino rats 62
Fig. 11 Effect of MEPA on serum urea of albino rats 64
Fig.12 Effect of MEPA on serum creatinine of albino rats 66
Fig.13 Effect of MEPA on serum sodium level of albino rats 68
Fig.14 Effect of MEPA on serum potassium level of albino rats 70
Fig.15 Effect of MEPA on serum Chloride level of albino rats 72
Fig.16 Effect of MEPA on packed cell volume (PCV) of albino rats 74
Fig. 17 Effect of MEPA on haemoglobin concentration of albino rats 76
Fig.18 Effect of MEPA on white blood cells count (WBC) of albino rats 78

LIST OF TABLES

Table 1 Qualitative Composition of Phyllanthus amarus 39
Table 2 Quantitative Composition of Phyllanthus amarus 40
Table 3 The percentage inhibition of DPPH radical by methanol extract of
Phyllanthus amarus (MEPA) 42
Table 4 Superoxide radical scavenging activity of MEPA 44
Table 5 Hydroxyl radical scavenging activity of MEPA 46
Table 6 The acute toxicity test result 48
Table 7 The summary of the histopathological examination of livers 87
Table 8 The summary of the histopathological examination of kidneys 89

LIST OF PLATES

Plate 1 Photomicrograph normal morphology of liver lobules of group 1 (control) 79
Plate 2 Photomicrograph of intact tubule content of the kidney of group 1 (control) 80
Plate 3 Photomicrograph of the liver of group 2 (100mg/kgbw) with extravasation of the
sinusoid and necrotic region 81
Plate 4 Photomicrograph of the kidney of group 2 (100 mg/kgbw) rat with elongated and shrunken proximal tubules. 82
Plate 5 Photomicrograph of the liver of group 3 rat showing apoptosis of the hepatocytes
and necrotic region. 83
Plate 6 Photomicrograph of the rat kidney in group 3 (200 mg/kgbw).
Shrunken proximal tubules and enlarged bowman’s capsule characterize the cells. 84
Plate 7 Photomicrograph of the liver of group 4 rat fed with 400 mg/kgbw. with
hypertrophy of hepatocytes and dead parenchyma cell observed. 85
Plate 8 Representing the photomicrograph of the kidney of group 4 rat with massive
densely clogged thick macula densa around the tubules (white arrow) and region of dead cells 86

LIST OF ABBREVIATIONS

DPPH 1,1-Diphenyl-2-picrylhydrazyl radical
EC50 Effective Concentration at 50% Inhibition
GFR Glomerular Filtration Rate
LD50 Median Lethal Dose
LDL Low Density Lipoprotein
ROS Reactive oxygen species
LOOH Lipid peroxide
LOO- Lipid peroxyl radical
NBT Nitro blue tetrazolium
MEPA Methanol extracts of Phyllanthus amarus
OH- Hydroxyl radical
H2O2 Hydrogen Peroxide
NO- Nitric oxide radical
O2- Superoxide radical
O2-1 Singlet oxygen
O3 Ozone
OS Oxidative stress
RO2- Peroxyl radical
U/L Unit/litre

CHAPTER ONE

INTRODUCTION

Plants have been the basis of many traditional medicine systems throughout the World for thousands of years and still remain as the main new source of structurally important chemical substances that lead to the development of innovative drugs (Fabricant and Farnsworth, 2001; Jachak and Saklani, 2007). The use of medicinal plants for the treatment of many diseases is associated with folk medicine from different parts of the World (Harvey, 2000 ; Bakhotmah and Alzahrani, 2010). Man therefore has a high dependency on plants which leads to its incorporation into their various ways of maintaining survival and livelihood including healthcare. For these reasons, the health of an average African depends more on his flora environment than the services of the orthodox physician located at a substantial geometrical separation from him (Ajibade et al., 2004).

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