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 Ë‚ 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 Ë‚ 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 Ë‚ 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 Ë‚ 0.05) level in all groups except group four in week one. In week two and three, there was a significantly higher level (p Ë‚ 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 Ë‚ 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 Ë‚ 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 Ë‚ 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 Ë‚ 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 Ë‚ 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         Â
Certification ii
Dedication iii
Acknowledgement                                                                                            iv
Abstract v
Table of Contents vi
List of Figures                                                                                           xii
List of Tables                                                                                            xiii     Â
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        Â
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
2.1.1Â Â Â Plant Materials (Phyllanthus amarus)Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 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                  Â
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)Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 19
2.2.3.5 Saponins                                                                                    19
2.2.3.6 Glycosides (Fehling’s Test)                                                    20                  Â
2.2.3.7 Reducing sugar                                                                       20
2.2.3.8 Â Â Â Â Â Â Â Â Â Â Â Tannins (Ferric Chloride)Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 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
2.2.10.1Â Â Â Â Â Â Â Â Â Â Packed Cell Volume (PCV)Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 35
2.2.10.2 Haemoglobin Estimation                                                                35
2.2.10.3 Total White Blood Cell (WBC) Count                                    36      Â
2.2.11 Histological Examination                                                      36      Â
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      Â
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      Â
3.8      Antioxidant Vitamin Contents of  MEPA                                          45
3.9      Acute Toxicity                                                                                        46      Â
3.10     Effect of MEPA on In Vivo Catalase Activity                             47
3.11    Effect of MEPA on Serum Alanine Aminotransferase (ALT) Activity 48
3.12Â Â Â Â Effect of MEPA on Serum Aspartate Aminotransferase (AST) Activity 49Â Â Â Â Â Â Â
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
3.24Â Â Â Â Histopathological Examination on the 100mg/Kg bw; Liver (Group Two)Â Â Â Â Â 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
REFERENCESÂ Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 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. 7Â Â Bar chart showing the in vivo catalase activity of MEPAÂ Â Â Â Â Â 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 4Â Â Â Â Â Â Â Â Â Â Â Superoxide radical scavenging activity of MEPAÂ Â Â Â Â Â Â Â Â Â Â Â Â Â 44
Table 5Â Â Â Â Â Â Â Â Â Â Â Hydroxyl radical scavenging activity of MEPAÂ Â Â Â Â Â Â Â Â Â Â Â Â 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).
