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PROJECT TOPIC AND MATERIAL ON Garcinia kolaHeckel STEM BARK ETHANOLIC EXTRACT AND ITS TRITERPENOID FRACTION PROTECTED AGAINST SODIUM ARSENITE-INDUCED HEPATOTOXICITY AND NEPHROTOXICITY IN RAT MODELS

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  • Name: Garcinia kolaHeckel STEM BARK ETHANOLIC EXTRACT AND ITS TRITERPENOID FRACTION PROTECTED AGAINST SODIUM ARSENITE-INDUCED HEPATOTOXICITY AND NEPHROTOXICITY IN RAT MODELS
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ABSTRACT

Arsenite is an environmental toxicant known to elicit adverse effects on liver and kidney organs. This study was designed to investigate the protective effects of Garcinia kola Heckel stem bark ethanolic extract (EEGK) and triterpenoid fraction (TFGK) against sodium arsenite-induced hepatotoxicity and nephrotoxicity in rats.

Sodium arsenite was used to induce hepatotoxicity and nephrotoxicity in Wistar strain albino rats for 14 days.EEGK and TFGK were used as test samples while silymarin served as a standard drug for comparison. Biomarkers measured were plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea, and creatinine. Ferric reducing antioxidant potential (FRAP), 1-1- diphenyl 2-picryl hydrazyl (DPPH), hydroxyl radical scavenging activity (HRSA), and total antioxidant capacity (TAC) assays were used to determine the antioxidant activity in vitro and In vivo antioxidant assays on the liver, kidney, and plasma superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) were carried out. In vitro mitochondrial membrane permeability transition (MMPT) was carried out. Histopathological examination of liver and kidney sections were performed and GC-MS analytical method was used to identify the bioactive compounds present in TFGK and EEGK.

Data showed that TFGK reduced ALT, AST, ALP activity and total bilirubin while EEGK reduced plasma creatinine and urea. Furthermore, EEGK elevated DPPH and hydroxyl radical scavenging activity, FRAP, and TAC when compared with TFGKin vitro. In addition, EEGK elevated plasma, liver and kidney SOD, GPx, GSH while TFGK modulated hematological markers. Further study showed thatTFGK inhibited the formation of liver and kidney MMPT.Histopathological examination showed that TFGK and EEGK reversed sodium arsenite-induced hepatotoxicity and nephrotoxicity respectively. GC/MS analysis detected 14 bioactive compounds in EEGK and 15 bioactive compounds in TFGK.

The study concluded that TFGK substantially protected against sodium arsenite-induced hepatotoxicity than EEGK while EEGKsubstantially protected against sodium arsenite-induced nephrotoxicity than TFGK. In addition, this study provided scientific insight to account for the traditional use of G. kola stem bark extract in ethnomedical practice.

Keywords: Stem bark; antioxidant; protection; toxicity; liver; kidney; mitochondrial;                                histopathology

Word Count: 327

TABLE OF CONTENTS

Content                                                                                                                                Page

Title Page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Abstract                                                                                                                                  v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          xii

List of Figures                                                                                                                         xiii

CHAPTER ONE: INTRODUCTION

1.1 Background to the Study                                                                                                 1

1.2 Statement of the Problem                                                                                                 2

1.3 Objective of the Study                                                                                                     2

1.4 Significance of the Study                                                                                                 3

CHAPTER TWO: REVIEW OF LITERATURE       

2.1Garcinia kola Heckel                                                                                                         4

2.2 Taxonomic classification                                                                                                   7

2.3 Scientific Research on G. kola                                                                                          7

2.3.1 Chemical constituents                                                                                                    7

2.3.2 Phytochemical constituents                                                                                           7

2.3.3 Hepatoprotective activity                                                                                              8

2.3.4 Analgesic effect                                                                                                             8

2.3.5 Histopathological Evaluation                                                                                         8

2.3.6 Anti-diabetic properties                                                                                                 8

2.3.7 Antiplasmodial, α-Glucosidase and Aromatase Inhibitory Activities                           8

2.3.8 Hepatoprotective and antioxidant activities                                                                  8

2.3.9 Anti-ulcer potential and proton pump inhibitory activity of G. kola seeds                   9

2.4Phytochemicals                                                                                                                  9

2.4.1 Biological Importance of Phytochemicals                     `                                               10

Content                                                                                                                                   Page

2.4.2 Classification of Phytochemicals                                                                                   10

2.4.2.1 Alkaloids                                                                                                                     10

2.4.2.2 Flavonoids                                                                                                                  11

2.4.2.3 Tannins                                                                                                                        13

2.4.2.4 Glycosides                                                                                                                  14

2.4.2.5 Phenolics                                                                                                                     15

2.4.2.6 Saponins                                                                                                                      16

2.4.2.7 Terpenoids                                                                                                                  17

2.5Antioxidants                                                                                                                      18

2.6Reactive Oxygen Species and Oxidative Stress                                                                18

2.7Gas Chromatography/Mass Spectrometry (GCMS) Analysis                                           20

2.8Hepatotoxicity                                                                                                                   21

2.8.1 Hepatotoxic Agents                                                                                                       22

2.8.2 Arsenic                                                                                                                           22

2.8.3 Mechanism of Action                                                                                                    23

2.9Liver                                                                                                                                   24

2.10Silymarin                                                                                                                          24

2.11Liver Function Test                                                                                                          25

2.11.1 Alanine Aminotransferase (ALT)                                                                                25

2.11.2 Aspartate Aminotransferase (AST)                                                                             25

2.11.3 Alkaline Phosphatase   (ALP)                                                                                      26

2.11.4 Bilirubin                                                                                                                       26

2.12Kidney                                                                                                                             27

2.13Kidney Function Test                                                                                                      28

2.13.1 Serum creatinine                                                                                                          28

2.13.2 Serum Urea                                                                                                                  28

2.14Mitochondria                                                                                                                   29

2.14.1 Mitochondrial Permeability Transition Pore                                                                30

 

 

Content                                                                                                                                   Page

CHAPTER THREE: METHODOLOGY

3.1 Materials                                                                                                                           32

3.1.1 Collection and identification                                                                                         32

3.1.2 Chemicals and reagents                                                                                                 32

3.1.3 Equipment                                                                                                                      32

3.2 Methods                                                                                                                            33

3.2.1 Preparation of plant extracts                                                                                          33

3.3 Quantitative phytochemical screening                                                                              34

3.3.1 Determination of total phenolic content (TPC)                                                             34

3.3.2 Determination of total flavonoid content (TFC)                                                           35

3.3.3 Determination of tannin concentration                                                                          35

3.3.4 Determination of saponin concentration                                                                        35

3.3.5 Determination of alkaloid concentration                                                                       36

3.4 Gas Chromatography/Mass Spectrometry (GC/MS) Analysis                                         36

3.5In vitro antioxidant assays for antioxidant property of G. kola stem bark                        36

3.5.1 Determination of DPPH (1-1-diphenyl 2-picryl hydrazyl) radical                                                        scavenging activity                                                                                                            36

3.5.2 Total Antioxidant Capacity (Phosphomolybdate assay)                                               37

3.5.3 Hydroxyl radical scavenging assay (HRSA)                                                                 37

3.5.4 Ferric Reducing Antioxidant Power (FRAP)                                                                38

3.6 Animal study                                                                                                                    38

3.6.1 Animal                                                                                                                           38

3.6.2 Acute Toxicity Test                                                                                                       38

3.6.3Experimental Design                                                                                                       39

3.7 In vivo antioxidant assays                                                                                                 40

3.7.1 Determination of reduced glutathione (GSH)                                                               40

3.7.2 Determination of Superoxide dismutase (SOD)                                                            41

3.7.3Determination of Glutathione Peroxidase (GPx)                                                           41

3.7.4 Lipid peroxidation                                                                                                         41

3.7.5 Protein                                                                                                                            41

Content                                                                                                                                   Page

3.8 Heamatological Assay                                                                                                      42

3.9Mitochondrial Assay                                                                                                          43

3.9.1Mitochondrial Permeability Transition                                                                           43

3.9.2 Preparation of low ionic strength rat liver mitochondria                                               43

3.9.3 Isolation of rat liver mitochondria                                                                                 43

3.10 Liver Function Test                                                                                                         45

3.10.1 Determination of Plasma Alanine Amino Transferase (ALT)                                     45

3.10.2 Determination of Plasma Aspartate Amino Transferase (AST)                                   46

3.10.3 Determination of Plasma Alkaline Phosphatase (ALP)                                               47

3.10.4 Determination of Total Bilirubin                                                                                 47

3.11Kidney Function Test                                                                                                      48

3.11.1 Serum Creatinine                                                                                                         48

3.11.2 Serum Urea                                                                                                                  49

3.12Histopathological Study                                                                                                  50

3.13Statistical Analysis                                                                                                           50

CHAPTER FOUR: DATA ANALYSIS, RESULTS AND        DISCUSSION OF FINDINGS

4.1Percentage yield of solvent fractions                                                                                 51

4.2Quantitative phytochemical analysis                                                                                 51

4.3 Acute Oral Toxicity                                                                                                          51

4.4In vitro antioxidant studies                                                                                                52

4.4.1 DPPH (1-1- diphenyl 2-picryl hydrazyl) radical scavenging activity                            52

4.4.2 Ferric Reducing Antioxidant Potential (FRAP)                                                                        54

4.4.3 Hydroxyl Radical Scavenging Assay (HRSA)                                                              55

4.4.4 Total Antioxidant Capacity (TAC)                                                                                56

4.5Liver Function Tests                                                                                                          57

4.5.1 Alanine aminotransferase (ALT)                                                                                   57

4.5.2 Aspartate aminotransferase (AST)                                                                                 58

4.5.3 Alkaline Phosphatase (ALP)                                                                                          59

 

Content                                                                                                                                   Page

4.5.4 Total Bilirubin                                                                                                                60

4.6Kidney Function Tests                                                                                                       61

4.6.1 Serum Creatinine                                                                                                           61

4.6.2 Serum Urea                                                                                                                    62

4.7In vivo antioxidant studies                                                                                     63

4.7.1 Reduced Glutathione (GSH)                                                                                         63

4.7.2 Glutathione Peroxidase (GPx)                                                                                       66

4.7.3 Superoxide Dismutase (SOD)                                                                                       69

4.7.4 Malondialdehyde concentration (MDA)                                                                       72

4.7.5 Protein                                                                                                                            75

4.8Heamotological Parameters                                                                                               78

4.8.1 White Blood Cell (WBC)                                                                                              78

4.8.2 Red Blood Cell (RBC)                                                                                                  79

4.8.3 Platelets                                                                                                                          80

4.8.4 Hemoglobin                                                                                                                   81

4.8.5 Hematocrit                                                                                                                     82

4.8.6 Neutrophils                                                                                                                    83

4.8.7 Lymphocytes                                                                                                                 84

4.8.8 Eosinophils, Monocytes and Basophils (EMB)                                                             85

4.9Mitochondrial Membrane Permeability Transition Assay (MMPT)                                   86

4.9.1 Assessment of liver MMPT                                                                                           86

4.9.2 Assessment of Kidney MMPT                                                                                      88

4.10 Gas Chromatography/ Mass Spectrometry (GC/MS) Analysis                                      90

4.11Histological Examination                                                                                     94

4.11.1 Histological examination of the liver                                                                           94

4.11.2 Histological examination of the kidney                                                                       95

CHAPTER FIVE: SUMMARY, CONCLUSION AND                                                                          RECOMMENDATIONS

5.1Summary                                                                                                                            98

5.2 Conclusion                                                                                                                        103

Content                                                                                                                                   Page

5.3 Recommendation                                                                                                              103

5.4 Contribution to Knowledge                                                                                              103

REFERENCES                                                                                                                104

APPENDICES                                                                                                                  124

 

  LIST OF TABLES

Table                                                                                                                                       Page

3.1 Distribution of five rats into nine groups                                                                             39

4.1 Quantitative phytochemical analysis of EEGK and TFGK                                                51

4.2 Acute oral toxicity testing of EEGK and TFGK                                                                51

4.3 Fifty percent inhibitory concentration (IC50) values for ascorbic acid,    EEGK                         and TFGK in different antioxidant models                                                                  53

4.4 Bioactivity of compounds detected through the GC/MS analysis of EEGK                      91

4.5 Bioactivity of compounds detected through the GC/MS analysis of TFGK                       92

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF FIGURES

Figure                                                                                                                          Page

2.1: The structure of the Garcinia bioflavonoids                                                                   5

2.2: G. kola plant from Ilishan Farm                                                                                       6

2.3: G. kolastem bark                                                                                                              6

2.4: Pharmacologically important plant alkaloids                                                                   11

2.5: Chemical structures of some flavonoids                                                                          12

2.6: Pharmacologically important flavonoids                                                                         13

2.7: Structural classification of Tannins                                                                                  14

2.8:Structural classification of some Glycosides                                                                    15

2.9: Pharmacologically important plantphenolics                                                                   16

2.10: Pharmacologically important planttriterpenoids                                                            18

2.11: Arsenic metabolism showing arsenate reduction to arsenite     and methylation                      to pentavalent                                                                                                             23

2.12: The proposed molecular mechanism of the mitochondrial permeability                        transition pore                                                                                                             31

3.1: Flow chart of the extraction of EEGK                                                                            33

3.2:Flow chart of the extraction TFGK                                                                                  34

4.1: DPPH radical scavenging activities ofEEGK and TFGK                                               52

4.2: Ferric reducing antioxidant potential of EEGK and TFGK                                            54

4.3:  Hydroxyl radical scavenging assay of EEGK and TFGK                                              55

4.4: Total antioxidant capacity of EEGK and TFGK                                                            56

4.5: Effects of varying doses of EEGK and TFGK   on alanine aminotransferase (ALT)                             activity in    arsenic-induced rats                                                                            57

4.6: Effects of varying doses of EEGK and TFGK on aspartate aminotransferase (AST)                activity in arsenic-induced rats                                                                            58

4.7: Effects of varying doses of EEGK and TFGK on alkaline phosphatase (ALP)                                     activity in arsenic-induced rats                                                                            59

4.8: Effects of varying doses of EEGK and TFGK on total bilirubin (mg/dL) levels in       arsenic-induced rats                                                                                                    60

 

Figure                                                                                                                                      Page

4.9: Effects of varying doses of EEGK and TFGK on serum creatinine (mg/dL) levels                   in arsenic-induced rats                                                                                          61

4.10: Effects of varying doses of EEGK and TFGK on urea levels (mg/dL) levels in          arsenic-induced rats                                                                                                    62

4.11: Effects of varying doses of EEGK and TFGK on plasma reduced glutathione levels                         in arsenic-induced rats                                                                                        63

4.12: Effects of varying doses of EEGK and TFGK on the liver reduced glutathione                     levels in arsenic-induced rats                                                                                        64

4.13: Effects of varying doses of EEGK and TFGK on the kidney reduced glutathione                 levels in arsenic- induced rats                                                                                        65

4.14: Effects of varying doses of EEGK and TFGK on plasma glutathione peroxidase                               levels in arsenic-induced rats                                                                                        66

4.15: Effects of varying doses of EEGK and TFGK on liver glutathione peroxidase                                   levels in arsenic-induced rats                                                                                        67

4.16: Effects of varying doses of EEGK and TFGK             on kidney glutathione peroxidase                   levels in arsenic-induced rats                                                                                        68

4.17: Effects of varying doses of EEGK and TFGK on plasma superoxide dismutase                                levels in arsenic-induced rats                                                                                        69

4.18: Effects of varying doses of EEGK and TFGK on liver superoxide dismutase                                    levels inarsenic-induced rats                                                                                     70

4.19: Effects of varying doses of EEGK and TFGK on kidney superoxidedismutase                     levels in arsenic-induced rats                                                                                        71

4.20: Effects of varying doses of EEGK and TFGKon plasma MDA levelsin arsenic-        induced rats                                                                                                                72

4.21: Effects of varying doses of EEGK and TFGK on liver MDA levels in arsenic-          induced rats                                                                                                                73

4.22: Effects of varying doses of EEGK and TFGK on kidney MDA levelsin arsenic-       induced rats                                                                                                                74

4.23: Effects of varying doses of EEGK and TFGK on plasma protein levels in arsenic-    induced rats                                                                                                                  75

Figure                                                                                                                                      Page

4.24: Effects of varying doses of EEGK and TFGK on liver protein levels in arsenic-        induced rats                                                                                                                  76

4.25: Effects of varying doses of EEGK and TFGK on kidney protein levels in arsenic-    induced rats                                                                                                                  77

4.26: Effects of varying doses of EEGK and TFGK on white blood cells in arsenic-          induced rats                                                                                                                  78

4.27: Effects of varying doses of EEGK and TFGK on red blood cells in arsenic-              induced rats                                                                                                                  79

4.28: Effects of varying doses of EEGK and TFGK on platelet counts in arsenic-              induced rats                                                                                                                  80

4.29: Effects of varying doses of EEGK and TFGK on hemoglobin levels in arsenic-                     induced rats                                                                                                                  81

4.30: Effects of varying doses of EEGK and TFGK on hematocrit levels in arsenic-                       induced rats                                                                                                                  82

4.31: Effects of varying doses of EEGK and TFGK on neutrophils count in arsenic-                      induced rats                                                                                                                  83

4.32: Effects of varying doses of EEGK and TFGK on lymphocytes count in arsenic-                   induced rats                                                                                                                  84

4.33: Effects of varying doses of EEGK and TFGK on EMB counts in arsenic-induced                rats                                                                                                                                85

4.34: Effects of  presence and absence of triggering agent Ca2+ on liver MMPT energized              sodium succinate and inhibited by spermine                                                                   86

4.35: Effects of varying concentrations of EEGK and TFGK on sodium arsenite-              induced liver MMPT energized by succinate for 12 mins                                         87

4.36: Effects of  presence and absence of triggering agent Ca2+ on kidney MMPT        energized sodium succinate and inhibited by spermine                                      88

4.37: Effects of varying concentrations of EEGK and TFGK on sodium arsenite-  induced kidney MMPT energized by succinate for 12 mins                              89

4.38:  GC-MS Chromatogram of EEGK of G. kola stem bark                                              90

4.39:  GC-MS Chromatogram of TFGK of G. kola stem bark                                               92

Figure                                                                                                                                        Page

4.40: Photomicrograph of the hepatic tissues of test animals                                                 95

4.41: Photomicrograph of the renal tissues of test animals                                                     97

CHAPTER ONE

INTRODUCTION

1.1 Background to the Study

Medicinal plants are a major source of phyto-compounds of beneficial values and are gaining countless significance in the essential wellbeing of individuals and social clubs in many nations. They  are  believed  to  be  nontoxic  and  utilized  in  the treatment  of  numerous diseases, nonetheless, concerns are drawn to lots of these plants due to their effectiveness, low noxiousness and absence / minimal adverse effects (Fawole et al., 2010). World Health Organization (WHO) has defined medicinal plants as plants that contain numerous properties that could be utilized for restorative purposes or those that manufacture metabolites to produce suitable drugs (WHO, 2008).

Contemporary research has been driven to investigate the effects of numerous medicinal plants that are believed to possess therapeutic properties for various body tissues, organs and systems. One of such plants that have gained much attention is Garcinia kola commonly called “bitter kola”. G. kola is normally consumed and used as remedy traditionally for different diseases (Ogunmoloye et al., 2012). It is vastly cherished for its therapeutic benefits due to its seed, stem and root serve as raw materials for pharmaceutical usage. The seed is ordinarily chewed as a masticatory in Nigeria to treat chest colds, cough, and liver disorders (Yakubu & Quadri, 2012), also as an emblem of amity and approval of guests (Otor et al., 2001). The stem bark is used in traditional medicine for the treatment of dysmenorrhea, inflammation and scorches (Iwu et al., 1990). The seed of G. kola possess anti-hepatotoxic, hypoglycemic, antioxidant, hypoglycemic and aphrodisiac properties (Akpanta et al., 2005) while the stem bark and root are soaked in local beers and taken orally for fever, cough, irritation and respiratory tract infections (Gil & Akinwunmi, 1986) .

Sodium arsenic (NaAsO2) is a toxic metallic pollutant of public health concern that is present in contaminated drinking water and ground water due to agricultural spill and mining process (Flora, 2004; Kapaj et al., 2006). In the environment, inorganic arsenic exist as arsenate (pentavalent, As5+) and arsenite (trivalent, As3+) and are readily interconvertible in aquatic environment through redox and methylation reactions. Chandranayagam et al. (2013) reported that sodium arsenite is sixty times stronger than sodium arsenate. Molecularly, arsenic is known to induce toxicity and carcinogenicity through the generation of oxidative stress and cell reactions as a result of the binding of arsenic to thiol (SH) groups of macromolecules (Tapio & Grosche, 2006). This binding results inalteration of several enzyme activities and proliferation of harmful reactive oxygen species (ROS) which prompts a wide array of heavy metal toxicities in human health (Shi et al., 2004). More so, the main targets of sodium arsenite induced toxicities are primarily the liver and kidneys. The most applied therapy for arsenite toxicity treatment has been metal chelation therapy which forms metal complexes with the consequent removal of excess arsenite from the body system (Chandranayagam et al., 2013). This type of therapy has not been without its adverse effects, however, the utilization  of plant extracts as a therapy against metal toxicity with minimal or no adverse effect could also be considered and scientifically validated.

1.2 Statement of the Problem

Arsenic poisoning has been treated with modern-day drugs including silymarin, meso 2,3-dimercaptosuccinic acid  (DMSA) and British Anti-Lewisite (BAL; 2,3-dimercaprol)  which are known to bring about antagonistic impacts to patients. Numerous conventional healers are known to utilize therapeutic plant extracts including garlic, curcumin and Moringa oleifera for the treatment of arsenic poisoning. However, there are little or no scientific information on the hepatoprotective and nephroprotective effects of G. kola stem bark against sodium arsenite-induced toxicity. Hence, there is a need to ascertain the therapeutic potency of G. kola stem bark extract utilized in the management of sodium arsenite-induced tissue toxicity.

1.3 Objective of the Study

The main objective is to investigate the hepatoprotective and nephroprotective effects of G. kola stem bark ethanolic extract and triterpenoid fraction against sodium arsenite-induced toxicity in rats.  The specific objectives are to:

  1. determine the phytochemical constituents in ethanolic extract and triterpenoid fraction of kola stem bark;
  2. determine the antioxidant activity of ethanolic extract and triterpenoid fraction of kola stem bark in vitro;
  3. investigate the effects of ethanolic extract and triterpenoid fraction of kola stem bark on liver function markers in rats exposed to sodium arsenite toxicity;
  4. investigate the effects of ethanolic extract and triterpenoid fraction of kola stem bark on kidney function markers in rats exposed to sodium arsenite toxicity;
  5. investigate the effects of ethanolic extract and triterpenoid fraction of kola stem bark on oxidative stress markers in rats exposed to sodium arsenite toxicity;
  6. investigate the effect of ethanolic extract and triterpenoid fraction of kola stem bark on sodium arsenite-induced mitochondrial membrane permeability transition in liver and kidney of rats in vitro and;
  7. examine the effects of ethanolic extract and triterpenoid fraction of kola stem bark on the histopathology of liver and kidney of rats exposed to sodium arsenite toxicity.

1.4 Significance of the Study

This investigation could contribute to the scientific basis on the ethnomedical utilization of G. kola stem bark in administration of sodium arsenite-induced liver and kidney toxicities. It could open up opportunities for further research into the improvement of G. kola stem bark bioactive compounds with a possibility of developing a new pharmaceutical drug.

 

 

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