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

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



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="" a="" administration="" an="" and="" anti-inflammatory="" aqueous="" cd="" compared="" control="" decrease="" delayed="" different="" effect="" ethanol="" extracts.="" extracts="" fractions="" given="" group="" groups.="" groups="" hypersensitivity="" immune-compromised="" immune-stimulating="" in="" increase="" investigated="" mean="" oedema="" of="" on="" oral="" p="" paw="" pcv="" rats="" reaction="" result="" results="" second="" showed="" significant="" span="" stage="" suggesting="" test="" the="" to="" twbc="" type="" untreated="" which="">4+ 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="" a="" acid="" activities="" and="" antioxidants="" as="" assay="" both="" calcium="" catalase="" compared="" compound="" concentration="" control.="" control="" cytokine="" decrease="" difference="" dismutase="" e="" expression="" extent="" extracts="" factor-alpha="" fraction="" gc-ms="" given="" glutathione="" group.="" groups="" hot="" hydroxy-4-methoxyphenyl="" i="" immune-compromised="" in="" increase="" increased="" interferon-gamma="" interleukin-10="" interleukin-2="" iron="" less="" level="" major="" malondialdehyde="" methanol="" most="" necrosis="" nmr="" no="" normal="" observed="" of="" on="" p="" peroxidase="" prop-2-enoic="" rats="" reduced="" reduction="" result="" results="" revealed="" selenium="" serum="" showed="" significant="" significantly="" similarly="" stimulation="" studies="" superoxide="" test="" the="" there="" to="" tumour="" untreated="" vitamin="" was="" water="" when="" zinc="">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.


CHAPTER ONE
INTRODUCTION
The reality of our modern society shows a preponderance of activities that elevate free radicals generation, engender stress, ultimately weaken the immune system and increase susceptibility to infections and diseases. The immune system is a system of biological structure and processes within an organism that protect against disease. It is designed to protect the host from invading pathogens and to eliminate disease (Sharmaet al., 2004; Naga and Rajeshwari, 2014).Immune system is core to maintenance of health and general well-being and is intricately associated with the four major causes of death which include injury, infection, degenerative disorders and cancer. Immunity is concerned with the recognition and disposal of foreign materials that enter the body while immunology is the study of how immune components respond and interact, of the consequences (desirable and otherwise), of their activity and of the ways in which they can be advantageously increased or reduced. There are two aspects of immune protection, the innate response and the adaptive response (Atal et al., 1986; Guyton and Hall, 2006). Innate immunity is present at birth, and provides the first barrier against infectious micro-organisms. Adaptive immunity is the second barrier against infections. It is acquired later in life and retains a memory of the invaders it has encountered (Nworu, 2007). Innate and adaptive mechanisms can be modified by substances to either enhance or suppress the ability to resist invasion by pathogens (Williams and Barclay1988).

The immune system is known to be involved in the etiology as well as the pathophysiologic mechanism of many diseases (Kalpeshet al., 2009). Immunology is thus probably one of the most rapidly developing areas of biomedical research holding great promise with regard to prevention and treatment of a wide range of disorders (Patilet al., 2012). Key elements of the immune response include recognition of self and non-self (Karlsen and Dryberg, 1998), regulation of immune response (Jerne, 1984); termination of immune response after effective control of offending agent (Parjis and Abbas, 1998) and establishment of a repertoire of memory cells for the future. The rise in immunological disorders confronting mankind today is alarming. This rise is due to different etiologies including environmental and nutritional habits. Disorders of the immune system include multiple sclerosis, arthritis, congestive heart failure, autoimmune disorders, several inflammatory disorders and infectious diseases such as AIDS, malaria, typhoid fever and the most dreaded Ebola virus disease. Immune function disorder is responsible for these and other diseases (Patwardhan et al., 1990). The immune system can be influenced by nutritional/metabolic status (Procaccini et al., 2013). Agents that alter the immune system either by stimulating or suppressing it are of great significance in managing.....

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