ACTIVATION OF CASPASE ACTIVITY IN RAT LIVER CELLS BY METHANOL EXTRACT OF GLORIOSA SUPERBA

Activation of caspase 3&9 by methanol extract of Gloriosa superba 

ABSTRACT

Mitochondrion is the key regulator in cellular energy homeostasis and plays a central role in determining cell apoptotic process; it is therefore regarded as a vital target for cancer chemotherapy. Cancer is obviously one of the most common areas where demand for alternative treatment is overwhelming. Many investigations revealed that bioactive compounds in natural products can act on mitochondria to trigger the permeabilization of the mitochondrial outer membrane and lead to the impairment of the mitochondria, including the alteration of electron transport, the loss of mitochondrial transmembrane potential, and the cytosolic release of apoptotic proteins such as Cytochrome C. Gloriosa superba is a medicinal plant used traditionally as anti-fungal, anti-bacteria and anti-carcinogenic agent. This study evaluates the modulatory effects of varying concentrations (10, 30, 50, 70 and 90µg/ml) and varying doses(50,100,200 and 400mg/kg) of methanol extract of Gloriosa superba on rat liver mitochondrial membrane permeability transition pore (mPT),as well as the release of caspases 9 and 3 which are the main regulators of mitochondria-mediated apoptosis.

Rats with approximately 100g weight were used for this experiment. Rat liver mitochondria were isolated by differential centrifugation, the mPT pore opening ,ATPase activity, lipid peroxidation, Cytochrome C release were assayed for at 540nm,660nm,532nm and 414nm respectively.

In the presence of calcium, there was induction of opening of the mPT pore which was reversed by spermine, a standard inhibitor. In the absence of calcium, methanol extract induced mPT pore opening in a concentration dependent manner. Mitochondrial ATPase activity was enhanced by the methanol extract with highest ATPase activity of 79% at 90µg/ml. The MEGS also inhibited Fe2+-induced lipid peroxidation with the highest inhibition at 50µg/ml. MEGS also induces Cytochrome C release at the highest concentration of 90µg/ml. 

The in-vivo analysis also indicated that the extract induced mPT pore opening of rat liver mitochondria in a dosage-dependent manner, with the highest induction at 400mg/kg. Oral administration of MEGS also indicated increase in the activation of caspases 9 and 3, the initiator and executioner of mitochondria-mediated apoptosis.

These findings suggest that the bioactive agents that induce pore opening are present in the methanol extract and thus may be used as drug candidates in situations where apoptosis up regulation is necessary.

TABLE OF CONTENTS

TITLE PAGE………………………………………………………………………………………I

CERTIFICATION II

DEDICATION III

ACKNOWLEDGEMENTS IV

TABLE OF CONTENTS V

LIST OF FIGURES IX

LIST OF TABLES. XII

ABSTRACT XIII

CHAPTER ONE I

INTRODUCTION AND LITERATURE REVIEW I

1.0 INTRODUCTION I

1. JUSTIFICATION IV
2. OBJECTIVES OF THE STUDY V
3. LITERATURE REVIEW VI

1.3.0 MITOCHONDRIA VI

1. STRUCTURE OF THE MITOCHONDRION VII

1.3.2 Energy Metabolism in Mitochondria XI

3. Heat Production XIV

1.3.4 Oxidative Phosphorylation XIV

1.4. MORPHOLOGY OF APOPTOSIS XXII

1.4.1 The Significance of Apoptosis XXIII

1.4.2 Distinguishing Apoptosis from Necrosis XXIV

1.4.3 Mechanisms of Apoptosis XXVIII

1.4.4 CASPASES: The Initiators and Executioners of Apoptosis XXXII

1.4.5 PERFORIN/GRANZYME PATHWAY XXXV

1.4.6 REGULATION OF APOPTOSIS BY IAPS XXXVII

1.4.7 INHIBITION OF APOPTOSIS XL

1.4.8 ASSAYS FOR APOPTOSIS XLI

1.4.9 APOPTOSIS IN DISEASE PATHOLOGY XLII

1.5 MITOCHONDRIA IN APOPTOSIS XLVI

1.5.1 MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION (mPT) LII

1.6 MEDICINAL PLANTS LX

1.6.1 Gloriosa superba LXI

CHAPTER TWO LXVII

2.1  PLANT MATERIAL LXVII

2.1.1  Collection of plant material LXVII

2.1.2 Preparation of extract LXVIII

2.1.3 Experimental animals LXVIII

2.2 PREPARATION OF LOW IONIC STRENGTH LIVER MITOCHONDRIA LXVIII

2.2.1 REAGENTS LXVIII

2.2.2 PROCEDURE LXIX

2.3 PROTEIN DETERMINATION LXX

2.3.1 PRINCIPLE LXX

2.3.2 REAGENTS LXXI

2.3.3 PREPARATION OF FOLIN-CIOCALTEAU REAGENT LXXI

2.3.4 STANDARD PROTEIN SOLUTION LXXII

2.3.5 PROCEDURE LXXII

2.3.6 SAMPLE PREPARATIONS LXXII

2.4 ASSESSMENT OF MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION IN RAT LIVER MITOCHONDRIA LXXV

2.4.1 PRINCIPLE LXXV

2.4.2 REAGENTS LXXV

2.4.3 PROCEDURE LXXVI

2.5 DETERMINATION OF MITOCHONDRIAL ATPase ACTIVITY LXXVIII

2.5.1  PRINCIPLE LXXVIII

2.5.2 REAGENTS LXXVIII

2.5.3 PROCEDURE LXXIX

2.5.4 DETERMINATION OF INORGANIC PHOSPHATE LXXX

2.6 DETERMINATION OF LIPID PEROXIDATION LXXXIII

2.6.1 PRINCIPLE LXXXIII

2.6.2 REAGENTS LXXXIV

2.6.3 PROCEDURE LXXXIV

CHAPTER THREE LXXXVI

3.1 EVALUATION OF THE EFFECT OF Ca2+ AND SPERMINE ON RAT LIVER MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION PORE. LXXXVI

3.1.1 INTRODUCTION LXXXVI

3.1.2 PROCEDURE LXXXVII

3.1.3 RESULT LXXXVII

3.1.4 CONCLUSION LXXXVIII

3.2 EVALUATION OF THE EFFECT OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF GLORIOSA SUPERBA ON RAT LIVER MITOCHONDRIA MEMBRANE PERMEABILITY TRANSITION PORE IN THE ABSENCE AND PRESENCE OF CALCIUM. LXXXIX

3.2.1 INTRODUCTION LXXXIX

3.2.2 PROCEDURE XC

3.2.3 RESULTS XC

3.2.4 CONCLUSION XCI

3.3 EVALUATION OF EFFECTS OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF Gloriosa superba ON RAT LIVER MITOCHONDRIA ATPase ACTIVITY. XCIII

3.3.1 INTRODUCTION XCIII

3.3.2 PROCEDURE XCIV

3.3.3 RESULT XCIV

3.3.4 CONCLUSION XCIV

3.4 EVALUATION OF EFFECTS OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF Gloriosa superba ON RAT LIVER MITOCHONDRIA CYTOCROME C RELEASE. XCVI

3.4.1 INTRODUCTION XCVI

3.4.2 PROCEDURE XCVI

3.4.3 RESULT XCVII

3.4.4 CONCLUSION XCVII

3.5 EVALUATION OF THE EFFECT OF VARYING CONCENTRATIONS OF METHANOL EXTRACT OF Gloriosa superba ON LIPID PEROXIDATION IN NORMAL RAT LIVER MITOCHONDRIA. XCIX

3.5.1 INTRODUCTION XCIX

3.5.2 PROCEDURE XCIX

3.5.3 RESULT C

3.5.4 CONCLUSION C

EXPERIMENT 6 CII

3.6 EVALUATION OF VARYING DOSES OF THE METHANOL EXTRACT OF Gloriosa superba ON RAT LIVER MITOCHONDRIA MEMBRANE PERMEABILITY TRANSITION PORE CII

3.6.1 INTRODUCTION CII

3.6.2 PROCEDURE CII

3.6.3 RESULTS CIII

3.6.4 CONCLUSION CIII

EXPERIMENT 7 CIV

3.7 INFLUENCE OF METHANOL EXTRACT OF Gloriosa superba ON CASPSES 9 AND 3 ACTIVATION USING ELISA TECHNIQUE. CIV

3.5.1 INTRODUCTION CIV

3.5.2 PROCEDURE CIV

3.5.3 RESULTS CV

3.5.4 CONCLUSION CV

CHAPTER 4 CVIII

4.1 DISCUSSION CVIII

4.2 CONCLUSION CX

REFERENCES. CXI

LIST OF FIGURES

Figure 1.3.1: Structure of the Mitochondrion

Figure 1.3.2: Electron transport sequence 

Figure 1.3.4: Oxidative phosphorylation

Figure 1.4: Hallmarks of the apoptotic and necrotic cell death process

Figure 1.4.3: Intrinsic mitochondria-mediated pathway 

Figure 1.4.5: Apoptotic pathways 

Figure 1.4.6: Mammalian IAP family members 

Figure 1.5: Displacement of IAPs from caspases by Smac/Diablo 

Figure 1.5.1: Mitochondria: promising targets for cancer chemotherapy

Figure 1.6: Gloriosa superba leaf

Figure1.6.1.2: Colchicine

Figure 2.3: Standard protein curve

Figure 2.5.4: Standard phosphate curve

Figure 2.6: MDA reaction in lipid peroxidation assay

Figure 3.1: Calcium-induced mitochondrial membrane permeability transition pore opening in normal rat liver mitochondria and its reversal by spermine

Figure 3.2: Effects of varying concentrations of MEGS on rat liver mitochondrial Membrane permeability transition pore in the absence of calcium

Figure 3.3: Effects of varying concentrations of MEGS on mitochondrial ATPase activity

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