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
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:
- determine the phytochemical constituents in ethanolic extract and triterpenoid fraction of kola stem bark;
- determine the antioxidant activity of ethanolic extract and triterpenoid fraction of kola stem bark in vitro;
- investigate the effects of ethanolic extract and triterpenoid fraction of kola stem bark on liver function markers in rats exposed to sodium arsenite toxicity;
- investigate the effects of ethanolic extract and triterpenoid fraction of kola stem bark on kidney function markers in rats exposed to sodium arsenite toxicity;
- investigate the effects of ethanolic extract and triterpenoid fraction of kola stem bark on oxidative stress markers in rats exposed to sodium arsenite toxicity;
- 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;
- 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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