EVALUATION OF RADIOPROTECTIVE EFFECT OF GONGRONEMA LATIFOLIO LEAF EXTRACT ON WHOLE-BODY IRRADIATED WISTAR ALBINO RATS

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TABLE OF CONTENTS

Title Page        –           –           –           –           –           –           –           –           i

Certification    –           –           –           –           –           –           –           –           ii

Acknowledgement      –           –           –           –           –           –           –           iii

Table of Contents          –           –           –           –           –           –           –           iv

Appendices     –         –           –           –           –           –           –           –           vii

List of Figures –    –           –           –           –           –           –           –           viii

List of Tables  –       –           –           –           –           –           –           –           ix

Abstract.         –           –           –           –           –           –           –           –           x

CHAPTER ONE: INTRODUCTION

1.1 Background of the study –              –           –           –           –           –           1

1.2 Objectives of the study        –           –           –           –           –           –           5

1.3 Significance of the study  –    –           –           –           –           –           –           6

CHAPTER TWO: LITERATURE REVIEW

2.1 Conceptual Review           –           –           –           –           –           –           7

2.1.1 Radiation           –           –           –           –           –           –           –           –           7

2.1.2 Source of exposure to ionizing radiation.   –           –           –           8

2.1.2.1 Naturally occurring ionizing radiation sources           –           –           8

2.1.2.2 Anthropogenic source –           –           –           –           –           –           9

2.1.3 Ionizing Radiation         –           –           –           –           –           –           –           9

2.1.3.1 X-ray and gamma interaction with matter      –           –           –           11

2.1.3.1.1Coherent Scattering –           –           –           –           –           11

2.1.3.1.2 Compton Scattering             –           –           –           –           12

2.1.3.1.3 The Photoelectric Effect      –           –           –           –           13

2.1.3.1.4 Pair Production        –           –           –           –           –           14

2.1.3.2 Interaction of charged particles with matter. –           –           –           14

2.1.3.2.1Alpha particle 2+)   –           –           –           –           –           14

2.1.3.2.2 Proton          –           –           –           –           –           –           15

2.1.3.2.3 Electron        –           –           –           –           –           –           18

2.1.3.2.4 Beta Particle ( )   –           –           –           –           –           19

2.1.3.3 Neutron interactions   –           –           –           –           –           21

2.1.4 Biological Effects of Ionizing Radiation on tissue/cells      –           21

2.1.4.1 Direct action in cell impairment by Radiation            –           –           22

2.1.4.2 Indirect action in cell damage by ionizing Radiation.            –           22

2.1.4.3 Organ response to ionizing radiation    –           –           –           –           23

2.1.4.4 Nine possible outcome when a cell is irradiated         –           –           25

2.2  Radioprotector     –           –           –           –           –           –           –           –           21

2.2.1 Mechanisms of action for radioprotector           –           –           –           27

2.3  Gongronema latifolio       –            –           –           –           –           27

2.4 Empirical Review       –           –           –           –           –           –           –           26

CHAPTER THREE:  DESIGN, MATERIALS AND METHODS       

3.1 Design –     –     –           –           –           –           –           –           –           33

3.1.2 Location of Study-      –           –           –           –           –           –           33

3.1.3 Target population                 –           –           –           –           –           33

3.1.4 Sample size-       –               –           –           –           –           –           33

3.1.5 Animal selection and handling    –           –           –           –           –           34

3..1.6 Source of data –   –           –           –           –           –           –           34

3.2 Materials   –              –           –           –           –           –           –           –           34

3.2.1 Gongronema latifolio collection and identification             –           35

3.2.2 Instruments / Equipment     —          –           –           –           –           35

3.2.3 Chemicals           –           –           –           –           –           –           –           –           35

3.3 Methods           –           –           –           –           –           –           –           –           36

3.3.1 Preparation of Gongronema latifolio (GL) extract-       –       –           36

3.3.2 Ethanol extraction of GL       –           –           –           –           –           36

3.3.3 Qualitative Phytochemical Analysis  of Gongronema   Latifolio- 36       

3.3.3.1 Test for tannins           –           –           –           –           –           –           36

3.3.3.2 Test for alkaloids        –           –           –                      –            –           37

3.3.3.3 Test for saponins         –           –           –           –           –           –           37

3.3.3.4 Test for flavonoids      –           –           –           –           –           –           37

3.3.3.5 Test for phenols          –           –           –                      –            –           37

3.3.4. Quantitative Phytochemical Analysis  of Gongronema latifolio 38

3.3.4.1 Determination of Tannins       –           –                      –            –           38

3.3.4.2 Determination of Alkaloids    –           –           –           –           –           39

3.3.4.3 Determination of Flavonoid   –           –           —          –           –           39

3.3.4.4 Determination of Polyphenol  –           –           –           –           –           40

3.3.4.5 Determination of Saponin       –           –           –           –           –           40

3.3.5 Experimental Protocols  –     –           –           –           –           –           41

3.3.6 Sample collection                   –           –           –           –           –           42

3.3.7 Extract dose selection       –           –           –           –           –                       43

3.3.8 Irradiation of rats            –           –           –           –           –           –           43

3.3.9 Determination of liver function tests, lipid peroxidation, and scavenge of free radical activities parameters-    –      –            47

3.3.9.1. Determination of Alkaline phosphatase (ALP)        –            –           47

  • Determination of Alanine Aminotransferase (ALT) –            –           48
    • Determination of Aspartate Aminotransferase (AST) –          48

3.3.9.4 Estimation of lipid peroxidation         –                      –            –           49

3.3.9.5 Determination of reduced glutathione activity           –           –           49

3.3.9.6 Determination of catalase activity      –           –           –           –           50

3.3.9.7 Determination of superoxide dismutase activity –      –           –           50

3.3.10 Statistical analysis        –           –           –           –         –           –           –       51

CHAPTER FOUR: RESULTS AND DISCUSSION

4.1 Phytochemical analysis result       –              –           –           –           –           53

4.2 Physical Observations       –           –              –           –           –           –           54

4.3 Biochemical parameters    –           –   –           –           –           –           –           55

4.3.1 ALP parameter   –           –               –           –           –           –           55

4.3.2: ALTparameter   –           –            –           –           –           –           56

4.3.3 ASTparameter    –           –           –           –           –           –           –           –           57

4.3.4 MDA parameter –           –           –           –           –           –           –           –           58

4.3.6 GSH  parameter –           –           –           –           –           –           –           –           60

  • CAT parameter           –           –           –           –           –           –           –           61

4.3.7 SOD parameter  –           –           –           –           –           –           –           –           62

4.4. Discussion            –           –       –           –           –           –           –           –           71

  • Limitations / Challenges   –           –            –           –           –           76

CHAPTER FIVE: CONCLUSION AND RECOMMENDATION

5.1 Conclusion            –           –            –           –           –           –           –           78

5.2 Recommendation  –              –           –           –           –           –           –           78

References      –           –           –         –           –           –           –           –           80

APPENDICES

Appendix A    –    –  Calculation of graded doses of radiation administered to rats in experimental groups – experimental control, pre-treatment and post- groups

Appendix B     –          –           Descriptive table

Appendix C    –           –           Multiple comparison table

Appendix D    –           –           Preparation of chemicals and Reagents for phytochemical analysis

LIST OF TABLES

Table 3.1 Design of the study             –           –           –           –           –           – 42    Table 4.1 Bioactive phytochemicals present in G. latifolio extract –     53 Table 4.2 Physical Observation   –   –           –           –           –           –           – 54 Table 4.3.1 ALP mean levelfor radiation doses and animal groups, before and after IR    55

Table 4.3.2 ALT mean level for radiation doses and animal groups, before and after IR   56 

Table 4.3.3 AST mean level for radiation doses and animal groups, before and after IR    57

Table 4.3.4 MDA mean level for  radiation doses and animal groups, before and after IR         58

Table 4.3.5 GSH mean level for radiation doses and animalgroups, before and after IR   60

Table 4.3.6 CAT mean level for radiation doses and animalgroups before and after IR       61

Table 4.3.7 SOD mean level for radiation doses and animalgroups before and after IR              62

LIST OF FIGURES

Figure 2.1 Frequencies of electromagnetic radiation and the corresponding photon energies and some of the applications for which they are used- –           –           –           –      7

Figure 2.2 Diagram of Rayleigh scattering        –                       –           –           12

Figure 2.3 Schematic Diagram of Compton scattering           –   –     13

Figure 2.4 Steps of cellular events that occur in the tissue after ionizing radiation exposure  –   –           –           –           –           –           –           25

Figure 2.5: Fresh leaves of Gongronema latifolio      –           –           –           44 Figure 3.1 a&b Wistar albino rats after acclimatization period   –      45 Figure 3.2 a&b Aligning of rat Immobilizer with the collimator field size of the LINAC with the help of laser light   –           –           –           –           –           46 Figure 3.3 a&b Positioning of LINAC gantry head for posterior Irradiation 47

Fig: 3.6 Oral administration of Gongronema latifolio extract –           48 Figure 4.1.1 Comparing mean body weight of rats in PRT and PST groups for 2Gy, 4Gy and 6Gy radiation dose respectively       –           –           –      63 Figure 4.1.2 Comparing ALP mean level in PRT and PST groups against NC group for  2Gy, 4Gy and 6Gy radiation doses respectively      –           –  64

Figure 4.1.3 Comparing ALT mean level in PRT and PST groups against NC group for  2Gy, 4Gy and 6Gy radiation doses respectively–           –      –    65

Figure 4.1.4 Comparing AST mean level in PRT and PST groups against NC group for  2Gy, 4Gy and 6Gy radiation doses respectively-           –            66

Figure 4.1.5 Comparing MDA mean level in PRT and PST groups against NC group for  2Gy, 4Gy and 6Gy radiation doses respectively- –      67

Figure 4.1.6 Comparing CAT mean activity level in PRT and PST groups against NC group for  2Gy, 4Gy and 6Gy radiation doses respectively    68

Figure 4.1.7 Comparing GSH mean activity level in PRT and PST groups against NC group for  2Gy, 4Gy and 6Gy radiation doses respectively    69

Figure 4.1.8 Comparing SOD mean activity level in PRT and PST groups against NC group for  2Gy, 4Gy and 6Gy radiation doses respectively    70

ABSTRACT

The purpose of the study was to evaluate the radioprotective effects of Gongronema latifolio (GL) leaf extract on a whole-body irradiated wistar albino rats. A prospective experimental and cross-sectional design was adopted for this study and it included a control group and experimental group. Part of the control group (normal control NC) was not irradiated neither was it administered with GL extract but the other part (experimental control EC) was only exposed to graded radiation doses (GRDs). In the experimental group, the pre-treatment group (PRT) received GL extract orally before being exposed to GRDs while post-treatment group were exposed to GRDs before receiving GL extract orally.

Phytochemical analysis of GL extract was done to re-determine the bioactive constituents of the extract. Physical changes were observed and recorded in all the groups using weight loss as an index. The blood samples of the animal groups were collected before and after irradiation (IR) for following analysis namely liver function test (LFT) {which includes-Alkaline phosphase (ALP), Alanine amino-transferase (ALT), Aspatate amino-transferase (AST)}, and antioxidant enzymes tests like Malondialdehyde (MDA), Glutathione (GSH), Catalase (CAT) and Superoxide dismutase (SOD)}.

The result of the phytochemical analysis revealed the presence of the following bioactive agents- alkaloids (3.11mg/g), tannins (2.43mg/g), flavonoids (1.31mg/g), phenols (1.10mg/g) and saponin (0.8mg/g). Body weight of the rats exposed to 6Gy in EC (51g) significantly (p<0.05) decreased when compared to NC (115g) and PRT (70g) but not significantly (p>0.05) different from PST (60g) group. ALP mean levels recorded in rats exposed to 4Gy increased (p<0.05) significantly in EC (74iU/L) when compared to PRT (37iU/L), PST (43iU/L) and NC (39iU/L) group on day 8 after IR. ALT mean level for rats exposed to 4Gy elevated (p<0.05) significantly in EC (50iU/L) relatively to PRT (31.67iU/L), PST (38.67iU/L) and NC (37iU/L) on day 8 after IR. MDA activity levels for rats exposed to 6Gy significantly (p<0.05) increased in EC (70%) relatively to PRT (35%), PST (59%) and NC (36%) on day 8 after IR. For rats exposed to 2Gy, GSH % activities decreased (p<0.05) significantly in EC (26%) when compared to PRT (59%) and NC (69%) on day 8 after IR. For rats exposed to 4Gy, CAT % activities significantly (p<0.05) decreased in EC (31%), PRT (49%) and PST (44%) relatively to NC (79%) on day 8 after IR. For rats exposed to 2Gy, SOD % activities decreased significantly in EC (29.33%), PRT (50.67%) and PST (40.67) when compared to NC (75%) on day 8 after IR.

Consequently, the result obtained suggested that GL extract emeroliates oxidative stress induced by ionizing radiation, thus affirming its radioprotective potentials. The result also demonstrated that the extract was more effective in PRT group relatively to PST group

CHAPTER ONE

INTRODUCTION

1.1 Background of the study

With the discovery of x-rays in 1895 and radioactivity in 1896, the biologic effects were also observed shortly after. Within the first six months of its use in treating patients, several cases of erythema, dermatitis and alopecia were already reported among x-ray operators and their patients. The first report of a skin cancer ascribed to x-rays was reported in 1902,  followed eight years later by experimental confirmation, Bushberget al., (2002).

Radiation medicine is one of the major sources of ionizing radiation due to its numerous applications in the hospital. Other sources of radiation exposure include radon in houses, contamination from weaponstesting sites, nuclear accidents and cosmic rays.Today, ionizing radiation is not only employed in treatment of diseases and industry but also in developing new varieties of high-yielding crops and enhancing storage period of food materials. Radiotherapy is one of the common sources of ionizing radiation and more so one of the most common modality used for treating human cancer. About 80% of cancer patients need radiotherapy at some time or the other either for curative or palliative purpose, Cherupally et al., (2001). It is essentially used in the treatment of a number of malignancies, but frequently its use is limited due to its adverse effects on normal tissue.

The effects of radiation on human cells/tissue can be divided into somatic and genetic effects. Somatic effects are harms exposed individual suffer during their life time such as radiation induced cancers, opacification of the eye etc, while genetic effects are radiation induced mutation to an individual genes and DNA that can contribute to the birth defective descendants Podgorsak, (2005). Somatic effects of radiation exposure can be classified as either stochastic or non-stochastic. A stochastic effect is the effect in which the probability of the effect, rather than its severity, increases with dose. Radiation-induced cancer and genetic effects are stochastic in nature. Stochastic effect is believed not to have a dose threshold. In non-stochastic effect, there is a threshold dose below which the effect is not seen. Cataract, erythema, fibrosis and hematopoietic damage are some of the non-stochastic effects that can result from large radiation exposure.

Radiation interactions that produce biologic changes are classified as either direct or indirect action. The change takes place by direct action if biologic macromolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or proteins become ionized or excited by an ionizing particle or photon passing through them or near them.The DNA damages caused per Gray are about 1000 single strand breaks (SSB), 40 double strand breaks and 950 base depurination. Roughly 4.4 x 107 single strand breaks, 1.4 x 107 double strand breaks and 1.1 x 107base lesion per year occur per mammalian cell, Fleck, et al,.(1999). Indirect effects are the result of radiation interactions within the medium (e.g. cytoplasm or water) which creates highly reactive free radicals chemical that in turn interact with the target molecule, Bushberget al., 2002). Because 70% to 85% of the mass of living system is composed of water, the vast majority of radiation-induced damage from medical irradiation is mediated through indirect action on water molecules. Exposure of biological tissues to ionizing radiation immediately leads to ionization and excitation of their constituent atoms. The molecules where the atoms reside then dissociate, resulting in so called free radicals, Mayles et al., (2007). This free radicals are reactive oxygen species such as hyoxyl radical (OH), superoxide radicals ( ), singlet oxygen and peroxyl radicals (ROO) in irradiated tissue that incite several pathophysiological changes in the body, Maurya, et al., (2011).

Free radicals can diffuse in the cell, producing damage at locations remote from their origin. They may inactive cellular mechanisms directly or via damage to genetic material (DNA and RNA), and they are believed to be the primary cause of biologic damage from low linear energy transfer (LET) radiation, Bushberg et al., (2002). It is estimated that two-thirds of DNA damage is caused indirectly by scavengeable radicals (Root and Okada, 1972), as reported in lobachevsky, et al., (n.d).Generally ionizing radiation causes either excitation or ionization or both to atoms and molecules which lead to the following conditions.

  • Generation of free radicals as mentioned earlier.
  • Breaking of chemical bonds.
  • Formation of new chemical bonds and cross-linkage between macromolecules.
  • Damage to biomolecules (e.g. DNA, RNA, Lipids, Proteins) which controls or regulates vital cell processes.

The detrimental consequences of irradiation (IR) of cells and tissues can be encountered in cancer radiation therapy. Apart from normal tissue damage, another issue associated with cancer radiotherapy is the potential for emergency of secondary radiation-induced cancers, affecting more than 1% of patients (Hall, 2006).Severally protective mechanisms have been adopted in radiotherapy to reduce oxidative stress in patients and it includes;

  • Physical protection (E.g. Conformal radiotherapy, intensity modulated radiotherapy IMRT etc).
  • Biological protection (E.g. hyperfractionation and Ultrafractionation).

Attempts have also been made to protect personnel working inradiation medicine departments, radiopharmaceutical centers, nuclear power operations, aviations, uranium miners and other sources of ionizing radiation through the provision of the following; personal dosimeter, shielding devices, radiation detection equipment and other safety procedures, policies etc so as to ensure safety of patient, occupational staff and the general public. But the truth is thationizing radiation and radioactive substances are natural and permanent features of theenvironment,and thus the risks associatedwith radiation exposure can only be restricted and cannot be eliminated entirely.

EVALUATION OF RADIOPROTECTIVE EFFECT OF GONGRONEMA LATIFOLIO LEAF EXTRACT ON WHOLE-BODY IRRADIATED WISTAR ALBINO RATS