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MODULATION OF IMMUNOLOGICAL RESPONSES IN ALBINO RATS BY LEAF EXTRACTS OF TELFAIRIA OCCIDENTALIS (HOOK F) AND TECTONA GRANDIS (LINN)

Bio Chemistry Project Topics & Materials

ABSTRACT

 

The immuno-modulating effects of leaf extracts of Telfairia occidentalis (Hook F) and Tectona grandis (Linn) on both humoral and cell mediated immune responses were evaluated in vivo. The responding cells were defined by flow cytometry and secretion of various cytokines by ELISA. Structural elucidation of the bioactive molecules responsible for the observed effect was equally attempted. Results of the quantitative phytochemical analyses of the extracts revealed abundance of bioactive compounds such as soluble carbohydrates (1.624 ± 0.002; 0.910 ± 0.003 mg/100g), tannin (6.593 ± 0.228; 5.325 ± 0.526 mg/1 00g), flavonoids (3.780 ± 0.228; 3.285 ± 0.526 mg/100g), saponins (3.285 ± 0.526; 0.744 ± 0. 004 mg/g), reducing sugars (293.364 ± 0.002; nil mg/100g), glycosides (8.683 ± 0.003; nil mg/g), terpenoids (2.436 ± 0.002; 2.546 ± 0.003 mg/100g), alkaloids (3.363 ± 2.247; nil mg/100g), phenol (8.574 ± 0.002; 8.096 ± 4.494 mg/100g) and hydrogen cyanide (0.395 ± 0.004; 0.344 ± 0.004 mg/g) for Telfairia and Tectona respectively. Acute toxicity studies carried out on the extracts showed no mortality or adverse reaction to the test mice up to a dose of 5000 mg/kg body weight which indicates that they are safe for consumption. The first stage of this study investigated the immune-modulating effect of aqueous and ethanol leaf extracts of Telfairia occidentalis and Tectona grandis on immune-compromised and non-immune-compromised rats. The results of packed cell volume (PCV), total white blood cell (tWBC) count, red blood cell (RBC) count, haemoglobin (Hb) concentration and humoral antibody titre showed a dose-dependent significant (p<0.05) increase in the groups given oral administration of aqueous and ethanol extracts compared to the untreated control groups. Result of the delayed type hypersensitivity (DTH) reaction showed a significant (p<0.05) decrease in mean paw oedema of rats in the test groups compared to the untreated immune-compromised group suggesting an anti-inflammatory effect of the extracts. In the second stage which investigated the immune-stimulating effect of different fractions on immune-compromised rats, the results showed a significant (p<0.05) increase in PCV, tWBC and CD4+ counts of different groups given varying doses of different fractions of the extracts compared to the untreated control. The increased production of CD4+ lymphocytes by the extracts confirmed their relevance in this study. The third stage studied the immune-modulating and antioxidant effect of both methanol fraction and hot water extracts of Telfairia and Tectona. Myelo-suppression by pyrogallol resulted in increased lipid peroxidation. Treatment with the extracts resulted in a significant (p<0.05) decrease in the concentration of malondialdehyde (MDA) in the test groups compared to the untreated control. Results of antioxidants assay showed a significant (p<0.05) increase in serum activities of catalase, glutathione peroxidase and to a less extent superoxide dismutase in the test groups compared to the untreated control group. The concentration of reduced glutathione was significantly (p<0.05) increased in the test groups compared to the untreated control. Similarly the serum concentration of iron, calcium, selenium and vitamin E increased significantly (p<0.05) when compared to the untreated control. There was no significant difference observed in the level of zinc compared to the untreated control. Result of the cytokine assay revealed a significantly (p<0.05) increased stimulation in the serum expression of interleukin-10 and tumour necrosis factor-alpha in both normal and immune-compromised rats given methanol fraction and hot water extracts compared to the untreated control. There was significant (P<0.05) reduction in the level of interleukin-2 and interferon-gamma in most test groups compared to the untreated control. GC-MS and NMR studies on the extracts showed (2E)-3-(3-hydroxy-4-methoxyphenyl) prop-2-enoic acid as the major compound in Tectona grandis while 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one and linoleic acid were found to be the bio-molecules responsible the observed effects in Telfairia occidentalis. The study has provided compelling evidence for an immune-modulatory effect of the extracts investigated. It also confirmed that this effect is mediated via action on cytokine expression and synergistic anti-oxidant activity and that moderate boiling does not affect this effect adversely. The two plant extracts performed similarly in most of the parameters determined.

 

 

TABLE OF CONTENTS

 

Title Page

Certification

Dedication

Acknowledgement

Abstract

Table of Contents

List of Figures

List of Tables

List of Abbreviations

 

CHAPTER ONE: INTRODUCTION

1.1 Introduction

1.1.1 Immuno-modulation

1.1.1.1 Immuno-stimulation

1.1.1.2 Immuno-suppression

1.2 Innate immune system

1.2.1 Humoral barriers to infection

1.2.1.1 Inflammatory response

1.2.2 Components of the innate immune system

1.2.2.1 Complement system

1.2.2.2 Leukocytes

1.2.2.3 Phagocytes

1.2.2.4 Neutrophils, Macrophages and Dendritic cells

1.2.2.5 Natural killer cells

1.3 Adaptive immune response

1.3.1 The lymphocytes

1.3.2 Helper T-cells

1.3.3 Killer T-cells

1.3.4 The B-cells

1.3.5 Structure and function of immunoglobulins

1.3.5.1 Basic immunoglobulin structure

1.3.5.2 Immunoglobulin production

1.3.5.3 Classes or isotypes of immunoglobulin

1.3.5.3.1 Immunoglobulin M

1.3.5.3.2 Immunoglobulin G

1.3.5.3.3 Immunoglobulin D

1.3.5.3.4 Immunoglobulin A

1.3.5.3.5 Immunoglobulin E

1.4 Cytokines

1.4.1 Class of cytokines

1.4.1.1 Chemokines

1.4.1.2 Interferons

1.4.1.3 Interferons-γ

1.4.1.4 Interleukins

1.4.1.4.1 Interleukin-2

1.4.1.4.2 Interleukin-10

1.4.1.5 Tumor necrosis factor

1.4.1.5.1 Tumor necrosis factor-alpha (TNF-α)

1.5 Pyrogallol

1.6 Antioxidants

1.6.1 Types of antioxidants

1.6.2 Classifications of antioxidants

1.6.2.2 Functions of antioxidants

1.6.2.3 Glutathione

1.6.3 Tocopherols and tocotrienols (vitamin E)

1.6.2    Antioxidant enzymes

1.6.3    Superoxide dismutase

1.6.4.2 Catalase

1.7 Lipid peroxidation

1.7.1 Lipid peroxidation and immune system

1.8 Mineral elements

1.8.1 Biochemistry and functions of some mineral elements

1.8.2 Calcium (Ca)

1.8.3 Iron (Fe)

1.8.4 Zinc (Zn)

1.9 Telfaira occidentalis (fluted pumpkin)

1.9.1Medicinal and nutritional properties of Telfairia occidentalis

1.10 Tectona grandis Linn (teak)

1.10.1 Medicinal importance of Tectona grandis

1.11 Statement of problem

1.12 Justification

1.13 Rationale

1.14 Aim of study

1.15 Specific objectives of the study

 

CHAPTER TWO: MATERIALS AND METHODS

2.1       Materials

2.1.1    Chemicals and Reagents

2.1.2    Equipment

2.1.3    Plant material

2.2       Methods

2.2.1    Extraction of plant materials

2.2.1.1 Aqueous extract

2.2.1.2 Methanol extract

2.2.1.3 Fractionation of the extract

2.2.2    Column and thin layer chromatographic separation

2.2.3    Acute toxicity (LD50) test of extracts

2.2.4    Proximate analysis of T. occidentalis and T. grandis

2.2.4.1 Moisture content

2.2.4.2 Crude fibre

2.2.4.3 Total ash

2.2.4.4 Crude fat

2.2.4.5 Crude protein

2.2.4.6 Carbohydrate

2.2.5    Qualitative phytochemical analysis of leaves of Telfairia occidentalis and Tectona grandis

2.2.5.1 Test for alkaloids

2.2.5.2 Test for flavonoids

2.2.5.3 Test for glycosides

2.2.5.4 Test for saponins

2.2.5.5 Test for tannins

2.2.5.6 Test for terpenoids and steroids

2.2.6    Quantitative phytochemical analysis of T. occidentalis and T. grandis

2.2.6.1 Alkaloid determination

2.2.6.2 Flavonoids determination

2.2.6.3 Steroids determination

2.2.6.4 Terpenoid

2.2.6.5 Tannin

2.2.6.6 Glycosides

2.2.6.7 Cyanogenic glycosides

2.2.6.8 Soluble carbohydrates

2.2.6.9 Reducing sugars

2.2.7    Animals

2.2.7.1 Antigen

2.2.8 Experimental design

2.2.8.1 First stage

2.2.8.2 Second stage

2.2.8.3 Third stage

2.2.8.4 Final stage

2.2.9    Preliminary screening of ethanol and aqueous extracts for immunomodulatory activity

2.2.9.1 Studies on delayed type hypersensitivity response (DTHR)

2.2.9.2 Studies on humoral antibody (HA) response

2.2.10  Haematological assay

2.2.10.1 Determination of erythrocyte count by haemocytometry

2.2.10.2 Determination of total leucocyte count by haemocytometry

2.2.10.3 Packed Cell Volume (PCV) estimation

2.2.10.4 Determination of Haemoglobin (Hb) concentration

2.2.10.5 Determination of CD4+ count

2.2.11Determination of enzymatic antioxidants

2.2.11.1 Estimation of superoxide dismutase

2.2.11.2 Estimation of catalase

2.2.11.3 Estimation of glutathione peroxidise

2.2.12 Non-enzymatic antioxidants

2.2.12.1 Estimation of reduced glutathione

2.2.12.2 Determination of selenium

2.2.12.2 Estimation of vitamin E (alpha tocopherol)

2.2.13  Estimation of extent of lipid peroxidation (malondialdehyde)

2.2.14.1 Serum calcium determination

2.2.14.2 Serum zinc determination

2.2.14.3 Serum iron determination

2.2.15 Determination of cytokines: IL-2, IL-10, TNF-α and IFN-γ

 

CHAPTER THREE: RESULTS

3.1       Phytochemical analyses of leaf extracts of Telfairia occidentalis and Tectona grandis

3.2       Proximate analysis on leaf extracts of Telfairia occidentalis and Tectona grandis

3.3       Acute Toxicity and Lethal Dose (LD50) Test

3.4       Effect of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on total white blood cell (TWBC) count of rats

3.5       Effect of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on packed cell volume (PCV) of normal and immune suppressed rats

3.6       Effect of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on red blood cell (RBC) count of normal and immune suppressed rats

3.7       Effect of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on haemoglobin (Hb) concentration of normal and immune suppressed rat

3.8       Effect of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on humoral antibody response (primary) of normal and immune suppressed rats

3.9       Effect of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on humoral antibody response (secondary) of normal and immune suppressed rats

3.10     Effect of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on delayed type hypersensitivity (DTH) reaction in normal and immune suppressed rats

3.11   Effect of crude ethanol extract and column fractions of Telfairia occidentalis

and Tectona grandis on total white blood cell (tWBC) count of immune suppressed rats

3.12     Effect of crude ethanol extract and column fractions of Telfairia occidentalis and Tectona grandis on packed cell volume (PCV) count of immune suppressed rats

3.13     Effect of crude ethanol extract and column fractions of Telfairia occidentalis and Tectona grandis on CD4 + count of immune suppressed rats

3.14     Effect of methanol and hot water extract of Telfairia occidentalis and Tectona grandis on lipid peroxidation (MDA) in normal and immune suppressed rats

3.15     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on catalase activity in normal and immune suppressed rats

3.16     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on superoxide dismutase (SOD) activity in normal and immune suppressed rats

3.17     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on glutathione peroxidase (GPx) activity in normal and immune suppressed rats

3.18     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on reduced glutathione (GSH) concentration in normal and immune suppressed rats

3.19     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on vitamin E (Vit E) concentration in normal and immune suppressed rats

3.20     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on selenium concentration in normal and immune suppressed rats

3.21     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on calcium ion concentration in normal and immune suppressed rats

3.22     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on serum iron concentration in normal and immune suppressed rats

3.23     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on serum zinc ion concentration in normal and immune suppressed rats

3.24     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on serum interleukin-2 (IL-2) concentration in normal and immune suppressed rats

3.25     Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on serum interleukin-10 (IL-10) concentration in normal and immune suppressed rats

3.26     Effect of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on concentration of tumour necrosis factor-alpha (TNF-α) in normal and immune suppressed rats

3.27     Effect of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on concentration of interferon-gamma (IFN-γ) in normal and immune suppressed rats

3.28     Results of IR, GC-MS and H NMR analysis on methanol fraction of Tectona grandis and Telfairia occidentalis

CHAPTER FOUR: DISCUSSION

4.1       Discussion

4.2       Conclusion

4.3       Contribution to Knowledge

4.4       Recommendations for further research

References

Appendices

 

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