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

0
4

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).

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