Evaluation of the Immunomodulatory Activities of the Aqueous Extract and the Beta–D-Glucan-Rich Polysaccharide Fraction of Pleurotustuberregium(Pleurotaceae)

ABSTRACT Mushroom cuisines are global delicacies and have been used for decades, not only as food, but mostly for the health benefits which are claimed or scientifically proven. The present study explored the immunomodulatoryactivites of the hot aqueous extract of a local oyster mushroom, Pleurotustuberregium (Fr.) Singer (Pleurotaceae) (PT) and its β–D-Glucan-Rich Polysaccharide fraction (BGP).The effects of the aqueous extract (PT) and the β–D-Glucan-Rich Polysaccharide fraction (BGP) were tested on some specific and non-specific immune responses in immunecompetent mice and in culture of RAW 264.7 macrophage cells. The effect of the PT and BGP on specific cell mediated immune response was investigated by the delayed type hypersensitivity response (DTHR) whilethe effect of the extract on specific humoral immune responses of treated mice was determined by prime-boost immunization protocol using ovalbumin as antigen and followed by the determination of antibody titers in the sera using the enzyme-linked immunosorbent assay (ELISA).Their effect on non–specific immune responses was determined by the colloidal carbon-clearance assay in mice. The effect of BGP on functional maturation and activation of the monocytic cells was also determined by measuring the level of tumour necrosis factor (TNF)-α and inducible oxygen (iNO) expressed into culture supernatant using cytokine capture ELISA and Griess reagent respectively.Short-term oral administration of PT (100, 200 and 400mg/kg) or BGP (100 and 200mg/kg) elicited a dose-related increase in DTHR and increased the mean phagocytic clearance of colloidal carbon in mice as much as 7 – 10 fold when compared to the clearance in the untreated group of mice. In a homologous prime-boost immunization schedule, oral supplementation with PT (100, 200 and 400mg/kg) or BGP (100 and 200mg/kg) significantly (P

TABLE OF CONTENTS

TITLE PAGE - - - - - - - - - - i

CERTIFICATION - - - - - - - - - iii

DEDICATION - - - - - - - - - iv

ACKNOWLEDGMENTS - - - - - - - - v

ABSTRACT - - - - - - - - - vi

TABLE OF CONTENTS - - - - - - - - vii

LIST OF FIGURES - - - - - - - - - xi

LIST OF TABLES - - - - - - - - - xiii

LIST OF APPENDICES - - - - - - - - xiv

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW

1.1 Scientific Background- - - - - - - 1

1.2 Overview of the Immune System - - - - - 3

1.2.1 Organs of the Immune System - - - - - 3

1.2.1.1 Primary Lymphoid Organs - - - - - 3

1.2.1.2 Secondary Lymphoid Organ - - - - - - 5

1.2.2 Cells of the Immune System - - - - - 6

1.2.2.1 Lymphoid Stem Cells - - - - - - - 9

1.2.2.2 Myeloid Stem Cells - - - - - - - 11

1.2.3 Arms of the Immune System - - - - - - 14

1.2.3.1 Innate Immunity - - - - - - - 14

1.2.3.2 Adaptive Immunity - - - - - - 23

1.2.4 Overview of Antibodies - - - - - - 26

1.2.4.1 Classes of Immunoglobulin - - - - - 28

1.2.4.2 Roles of Antibodies - - - - - - - 29

1.3 Mediators of the Immune System - - - - - 31

1.3.1 Cytokines - - - - - - - - 31

1.3.2 Complement System - - - - - 35

1.4 Disorders of the Immune System - - - - - 38

1.4.1 Hypersensitivity - - - - - - - 38

1.4.2 Immune Deficiency Diseases - - - - - - 39

viii

1.4.3 Autoimmune Diseases - - - - - - 41

1.4.4 Graft Versus Host Diseases - - - - - - 42

1.4.5 Immune Complex Diseases - - - - - - 42

1.5 The Concept of Immunomodulation, Immunosuppression, Immunostimulation and

Immunotolerance - - - - - - - 43

1.5.1 Immunostimulation - - - - - - - 43

1.5.2 Immunosuppression - - - - - - - 44

1.5.3 Tolerance - - - - - - - - 45

1.6 Potentials of Mushroom as Immunomodulatory Substance of Natural Origin45

1.7 Beta (B) Glucans - - - - - - - 48

1.7.1 Beta Glucans and the Immune System - - - - 48

1.7.1.1 Beta GlucanImmunostimulating Activity - - - - 49

1.7.1.2 Beta Glucan Increases Resistance to Infectious Challenge - - 50

1.7.1.3 Beta GlucanAnticarcinogenic Activity - - - - 51

1.7.1.4 Beta Glucan as Adjuvant to Cancer Chemotherapy and Radiotherapy 51

1.8 Botanical Profile and Review Of Pleurotustuberregium - - 52

1.8.1 Taxonomy of Pleurotustuberregium - - - - - 53

1.8.2 Botanical Description of Pleurotustuberregium - - - 53

1.8.3 Geographical Distribution of Pleurotustuberregium - - - 55

1.8.4 Ethnomedicinal and Folkloric Uses of Pleurotustuberregium - 55

1.8.5 Pharmacological Studies, Phytochemical and Proximate Constituents

ofP. tuberregium - - - - - - - 56

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials - - - - - - - - 57

2.1.1 Chemicals and Reagents - - - - - - 57

2.1.2 Equipment - - - - - - - - 57

2.2 Method - - - - - - - - 57

2.2.1 Collection and Authentication of Plant Materials - - - 57

2.2.2 Preparation of Plant Material - - - - - - 58

2.2.3 Bioactivity Guided Fractionation of Crude Extract - - - 58

2.2.4 Extraction of the β glucan rich Polysaccharide Fraction of

ix

Pleurotustuberregium - - - - - - - 60

2.2.5 Phytochemical Analysis of Extracts - - - - - 60

2.2.6 Pharmacological Studies - - - - - - 61

2.2.6.1 Animals - - - - - - - - 61

2.2.6.2 Antigens - - - - - - - - 61

2.2.6.3 Acute Toxicity (LD50) Test of Extracts - - - - 61

2.2.7 Studies on the Hot Aqueous Extract of Pleurotustuberregium(PT)

and the βGlucan Enriched Polysaccharide Fraction of Pleurotus

tuberregium(BGP) - - - - - - - 62

2.2.7.1 Studies on Relative Spleen Weight - - - - - 62

2.2.7.2 Studies on Phagocytic Index - - - - - - 62

2.2.7.3 Studies on Delayed Type Hypersensitivity Response (DTHR) - 63

2.2.7.4 Studies on Humoral Antibody Response Induced by Ovalbumin - 63

2.2.8 In vitro Studies - - - - - - - 65

2.2.8.1 Spleenocytes Proliferation Assay - - - - - 66

2.2.8.2 Effect of BGP on Inducible Nitric Oxide (iNO) Production/Release- 66

2.2.8.3 Effect of BGP on Tumour Necrosis Factor (TNF- Α)

Production /Release - - - - - - - 67

2.2.8.4 Effect of BGP on Rate of Phagocytosis of Macrophages - - 67

2.9.0 Statistical Analysis - - - - - - - 68

CHAPTER THREE: RESULTS

3.1 Extraction - - - - - - - - 69

3.2 Phytochemical Analysis of Extract and Fraction - - - 69

3.3 Acute Toxicity Test - - - - - - - 69

3.4 Effect of Aqueous Extract of Pleurotustuberregium on Phagocytic Activity in

Mice - - - - - - - - - 69

3.5 The Effect of Hot Aqueous Extract of P. tuberregium on the Relative Spleen

Weight ofMice - - - - - - - 73

3.6 The Effect of Pleurotustuberregium Extracts on Delayed Type Hypersensitivity

response (DTHR) - - - - - - - 75

3.7 The Effect of Hot aqueous extract (PT) and the β Glucan rich

x

Polysaccharide Fraction of Pleurotustuberregium(BGP)on Humoral

Antibody Synthesis Induced by Ovalbumin - - - - 78

3.7.1 The Effect of Hot of Aqueous Extract of Pleurotustuberregium on

Primary and Secondary Humoral Immune Responses to Ovalbumin

in Mice - - - - - - - - 78

3.8 Effect of Βeta (β)-Glucan rich Polysaccharide Fraction of

Pleurotustuberregium (BGP) on Primary and Secondary Humoral immune

Responses to Ovalbumin in Mice - - - - - 82

3.9 Invitro studies - - - - - - - - 89

3.9.1 Effect of Βeta (β)-Glucan rich Polysaccharide Fraction of P.

tuberregium (BGP) on Spleenocytes Proliferation - - - 89

3.9.2 Efect of BGP ON TNF-α Production by RAW264.7 - - - 89

3.9.3 Effect of BGP on Nitric Oxide Production by Raw264.7 - - 90

3.9.4 Effect of BGP on the rate of Phagocytosis (Neutral Red Uptake)

RAW264.7 - - - - - - - - 90

CHAPTER FOUR: DISCUSSION AND CONCLUSION

4.1 Discussion - - - - - - - - 95

4.2 Conclusion - - - - - - - - 103

References

Appendices