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

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 immunocompromised and non-immunocompromised 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 immunocompromised group suggesting an anti-inflammatory effect of the extracts. In the second stage which investigated the immune-stimulating effect of different fractions on immunocompromised 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 immunocompromised 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 biomolecules 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 antioxidant 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 Telfairia occidentalis (fluted pumpkin)

1.9.1 Medicinal 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

LIST OF FIGURES

Fig.1: The Complement cascade

Fig.2: The activation of T and B cells

Fig.3: The structure of pyrogallol

Fig.4: Lipid peroxidation and generation of free radicals

Fig. 5: Leaves of Telfairia occidentalis (Hook F)

Fig. 6: Leaves of Tectona grandis Linn

Fig. 7: Effects of aqueous and ethanol extracts of Telfairia occidentalis and Tectona grandis on total white blood cell count of normal and immune suppressed rats

Fig 8: Effects of aqueous and ethanol extracts of Telfairia occidentalis and Tectona grandis on packed cell volume of normal and immune suppressed rats

Fig. 9: Effects of aqueous and ethanol extracts of Telfairia occidentalis and Tectona grandis on red blood cell count of normal and immune suppressed rats

Fig. 10: Effects of aqueous and ethanol extracts of Telfairia occidentalis and Tectona grandis on haemoglobin (Hb) concentration of normal and immune suppressed rats

Fig.11: Effects of aqueous and ethanol extracts of Telfairia occidentalis and Tectona grandis on humoral antibody response (primary) of normal and immune suppressed rats

Fig. 12: Effects of aqueous and ethanol extract of Telfairia occidentalis and Tectona grandis on humoral antibody response (secondary) of normal and immune suppressed rat

Fig. 13: Effects of aqueous and ethanol extracts of Telfairia occidentalis and Tectona grandis on delayed type hypersensitivity (DTH) reaction in normal and immune suppressed rats

Fig 14: Effects of crude extract and fractions of Telfairia occidentalis and Tectona grandis on total white blood cell (tWBC) count of immune suppressed rats

Fig. 15: Effects of crude ethanol extract and column fractions of Telfairia occidentalis and Tectona grandis on packed cell volume (PCV) count of immune suppressed rats

Fig. 16: Effects of crude ethanol extract and column fractions of Telfairia occidentalis and Tectona grandis on CD4+ count of immune suppressed rats

Fig. 17: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on lipid peroxidation (MDA) in normal and immune suppressed rats

Fig. 18: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on catalase activity in rats

Fig. 19: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on superoxide dismutase (SOD) activity in normal and immune suppressed rats

Fig. 20: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on glutathione peroxidase (GPx) activity in normal and immune suppressed rats

Fig. 21: Effect of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on reduced glutathione (GSH) concentration in normal and immune suppressed rats

Fig. 22: Effect of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on vitamin E (Vit E) concentration in normal and immune suppressed rats

Fig. 23: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on selenium concentration in normal and immune suppressed rats

Fig. 24: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on calcium ion concentration in normal and immune suppressed rats

Fig. 25: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on serum iron ion concentration in normal and immune suppressed rats

Fig. 26: Effects of methanol and hot water extracts of Telfairia and Tectona on zinc ion concentration in rats

Fig. 27: 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

Fig. 28: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on concentration of interleukin-10 (IL-10) in normal and immune suppressed rats

Fig. 29: Effects of methanol and hot water extracts of Telfairia occidentalis and Tectona grandis on concentration of tumor necrosis factor-alpha (TNF-α) in normal and immune suppressed rats

Fig. 30: Effects 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

Fig. 31: Structure of the compound (2E)-3-(3-hydroxy-4-methoxyphenyl) prop-2-enoic acid isolated from Tectona grandis

Fig. 32: Structure of compounds 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one and linoleic acid isolated from Telfairia occidentalis

LIST OF TABLES   

Table 1: Result of qualitative phytochemical analyses on leaf extracts of Telfairia occidentalis and Tectona grandis

Table 2: Result of quantitative phytochemical analyses on leaf extracts of Telfairia occidentalis and Tectona grandis

Table 3: Result of proximate analyses of Telfairia occidentalis and Tectona grandis

Table 4: Result of acute toxicity and lethal dose (LD50) test

LIST OF ABBREVIATION

4-HNE 4-hydroxynonena

AIDS Acquired immune deficiency syndrome

AOAC Association of official analytical chemists

APC Antigen-presenting cells

ATPase Adenosine triphosphatase

BRM Biologic response modifier

CAT Catalase

CD Cluster of differentiation

CDR Complementarity Determining Regions

CMI Cell mediated immunity

CTL Cytotoxic T-lymphocyte

DC Dendritic cell

DNA Deoxyribonucleic acid

DTH Delayed type hypersensitivity

DTNB Dithio-bis-2-nitrobenzoic

EAF Ethyl acetate fraction

EF Ethanol fraction

GC-MS Gas chromatography mass spectroscopy

GPX Glutathione peroxidase

GR Glutathione reductase

GSH Reduced glutathione

GSSG Oxidized glutathione

H2O2 Hydrogen peroxide

HA Humoral antibody

Hb Haemoglobin

HDL High density lipoprotein

HIV Human immune virus

IFN-γ Interferon-gamma

Ig Immunoglobulin

IL Interleukin

INT Iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride

IR Infra red

KIR Killer cell immunoglobulin receptor

LD Lethal dose

LOOH Lipid hydroperoxide

MBL-MAP Mannose-binding lectin pathway

MDA Malondialdehyde

ME Methanol extract

MF Methanol fraction

MHC Major histocompatibility complex

MS Multiple sclerosis

NFE Nitrogen Free Extract

NK Natural killer

NMR Nuclear magnetic resonance

O2˙ Superoxide anion

OH- Hydroxyl radical

PAMP Pathogen-associated molecular patterns

PCV Packed Cell Volume

PRR Pattern recognition receptors

PUFA Poly-unsaturated fatty acids

RBC Red blood cell

RNA Ribonucleic acid

ROS Reactive oxygen species

SDS Sodium dodecyl sulfate

SOD Superoxide dismutase

SRBC Sheep red blood cell

T. grandis Tectona grandis

T. occidentalis Telfairia occidentalis

TBA Thiobarbituric acid

TCA Trichloroacetic acid

TCGF T-cell growth factor

TCR T-cell receptor

TH T-helper

TLC Thin layer chromatography

TLR Toll-like receptor

TNF-α Tumour necrosis factor-alpha

tWBC Total white blood cell

UV Ultraviolet

Vit E Vitamin E

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 microorganisms. 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 immunological disorders and are known as immune-modulators (Srivathsa, 2006). Modulation of immune responses to alleviate various diseases has been of interest for many years (Sharma et al., 2004). In HIV and other infectious diseases, stimulation/up-regulation of the immune system is a highly desired goal. Immunostimulatory therapy is now recognized as an alternative to conventional chemotherapy for a variety of disease conditions involving impaired immune response of the host (Upadhaya, 2007; Ganjuet al., 2003). Immuno-stimulators are known to support T-cell function, activate macrophages and granulocytes and complement natural killer cells apart from the production of various effector molecules generated by activated cells (Wagner et al., 2003). The function and the efficacy of the immune system may be influenced by many exogenous factors like food and pharmaceuticals, physical and psychological stress and hormones. An immune-modulator essentially helps to optimize immune function by normalizing the process and thereby maintaining balance. Immune regulation is a complex balance between regulatory and effector cells and any imbalance in immunological mechanism can lead to a disease condition (Sehraet al., 2008). In healthy individuals immune-stimulants are expected to serve as prophylactic or promotive agents by enhancing basal levels of immune response and in individuals with impaired immunity they act as immunotherapeutic agent (Agrawal and Singh, 1999).

The immune system of humans is intricately interwoven with oxidative processes in the body. High oxidative stress usually breaks down the immune system, precipitates radicals as well as severe diseases and this must be prevented (Halliwell, 1992). Studies have emphasized the therapeutic importance of plant-derived immunomodulators with antioxidant activities (Allam, 2009; Guo et al., 2009). Modulation of the immune system as well as optimizing oxidative processes of the body with aid of natural products represents a field of drug development-based research witnessing unprecedented upsurge in recent times (Nworu, 2007). A newer approach to therapeutics is to search for potent immune-modulating substances preferably with synergistic antioxidant activity. Indeed there has been growing interest in isolating and characterizing compounds with immunomodulatory and antioxidant activities (Wang et al., 2004). It has been established that most pharmacologic activities are related to the immune-modulatory and antioxidant activities of plant secondary metabolites (Okonji et al., 1999).

 

More than a quarter of medicines in use today come from plants and these medicinal plants serve as therapeutic alternatives, safer choices or in some cases as the only effective treatment (Sharififaret al., 2009). The unmatched availability and chemical diversity characterizing natural products provide unlimited opportunities for development of new drug leads (Cos et al., 2006). Natural products are still major sources of innovative therapeutic agents for various conditions including infectious diseases (Clardy and Walsh 2004). Increased interest in herbs has prompted scientific scrutiny of their therapeutic potential and safety (Atal et al., 1986). The use of products of plants and animal origin as medicinal agents is predicated upon the belief that they promote positive health and maintain organic resistance against infections by re-establishing the bodys̓equilibrium and conditioning of body tissues(Fulzele et al., 2003). Use of plant remedies again is perceived as a natural approach to disease treatment. Equally medicinal plants are rich sources of substances which are claimed to induce para-immunity (Koreet al., 2010) and relieve oxidative stress (Njoku and Adikwu, 1997). Phytochemical constituents such as terpenoids, steroids, proteins, tannins and flavonoids are considered responsible for immune-modulatory properties exhibited by plants (Kuo et al., 2004).Telfairia occidentalis Hook F and Tectona grandis Linn are well known for haematinic and other medicinal properties. However there is no documented evidence of any serious investigation of their immune-modulatory effects (Kayode and Kayode, 2010). The search for safe and potent immune-modulating substances preferably with synergistic antioxidant activities continues to attract great research interest (Wang et al., 2004). There is no doubt that immune-modulators hold great promise for control and prevention of infections but is yet to be fully exploited (Nworu, 2007).

1.1.1 Immuno-modulation

Immuno-modulation is a process which alters the immune system of an organism by interfering with its functions (Agrawal, 2010). An immuno-modulator therefore is a substance, biological or synthetic which can stimulate, suppress or modulate any of the components of the immune system. This could result in immuno-stimulation/enhancement or immuno-suppression (Dhasarathan et al., 2010; Vinotha Pooshan and Sundar, 2011). Immuno-modulators alter the immune system by interacting with it to up-regulate or down-regulate specific aspect of the host response (Stanilova et al., 2005; Utoh-Nedosa et al., 2009) they are of great importance in treating immunological disorders (Srivathsa, 2006). They are also known as biologic response modifiers or immunoregulators which function as drugs, leading predominantly to a non-specific stimulation of immunological defense mechanisms (Tzianabos, 2010). Regulation of the immune response by an immune regulator is a normalization process that helps to optimize the immune response (Sehra et al., 2008; Agrawal, 2010). Immunomodulators may include some bacterial products, lymphokines and plant derived substances. The effects of immunomodulators can be classified into three which are stimulation, suppression and restoration of the immune system. Unlike vaccines, most immunomodulatory agents are not real antigens but antigen mimetics or so-called mitogens. They do not stimulate the development of memory lymphocytes, thus the effect of immunomodulatory agents towards specific immune system will be reduced after a short of period of time (Wagner and Fintelman, 1999). The ability of immunomodulators to enhance or suppress immune responses can depend on a number of factors such as dosage, route of administration, timing and frequency of administration (Tzianabos, 2010). Immunomodulation generally entails the adjustment of the immune system to the desired level and could be achieved by the use of natural as well as synthetic agents from plants and chemicals respectively. The immunomodulatory effect of plants can be explained most preferably using two scenario:immunostimulation and immunosuppression.

1.1.1.2 Immunostimulation

Immuno-stimulation is a process that involves the activation of the immune system activity and that of its components. Immunostimulatory agents are used often to achieve this purpose and are grouped into specific and nonspecific immune stimulants. Biologic response modifiers (BRM) are substances that stimulate the body’s response to infection and disease. The body is known to produce these substances in inappreciable quantity hence the need for exogenous supplementation from diets and pharmaceuticals. Immunostimulators are known to support T-cell function, activate macrophages, granulocytes and complement natural killer cells apart from affecting the production of various effector molecules generated by activated cells (Wagner et al., 2003). Immunostimulatory therapy has been long recognized as alternative to conventional chemotherapy for a variety of disease conditions involving the impaired immune response of the host (Ganju et al., 2003).

1.1.1.3 Immunosuppression

Immunosuppression is a component of immune modulation and involves the reduction of the activation or efficacy of the immune system. It also involves altering the sensitivity of the immune system and this can be achieved by the use of immuno-suppressive agents. Some portions of the immune system itself have immuno-suppressive effects on other parts and immunosuppression may occur as an adverse reaction to treatment of other conditions. Cytokines have been found to preferentially suppress or stimulate the production of immune cells depending on the prevailing conditions (Liu et al., 2013).The immune system can be manipulated to suppress unwanted responses resulting from autoimmunity and allergy. It therefore functions by reducing the effectiveness of the immune systems response to foreign substances. Other substances that induce immune suppression include rapamycin, pyrogallol and cyclophosphamide (Diken et al., 2013).

In general, immunosuppression can be induced in some special cases such as in organ transplant to prevent graft rejection and in treating autoimmune diseases.

1.2 Innate Immune System

The innate immunity is present at birth and does not require specific recognition of an antigen by the immune system (Vollmar, 2005). It is the first line of defense and is primarily mediated by various kinds of natural barriers including physical, chemical and enzymatic barriers. Innate immunity is involved in both humoral and cellular arms of immune response, to protect the host against a vast and diversified range of microbes and their products. The humoral effectors of innate immunity consist of families of soluble proteins such as complement and acute-phase response proteins. These are not only important for the initial neutralization or elimination of microbes, but also alert the host’s immunity through the recruitment of a variety of cells at the site of infection of tissue injury. The cellular arm consists of “nonprofessional” somatic cells such as epithelial cells and “professional” immune cells such as various types of tissue phagocytic cells and dendritic cells (Himanshu and Adrian, 2013).The cells of the innate immune system such as dendritic cells (DCs), detect and respond to pathogens through the expression of pattern recognition receptors (PRRs) including Toll-like receptors (TLRs), Nod-like receptors, and Dectin-1(Manicassamy and Pulendra, 2009; Takeuchi and Akira, 2010). PRRs bind to β-1,3-glucans, such as curdlan, on the cell wall of the fungi and some bacteria (Brown et al., 2002), thereby activating DCs (Kennedy et al., 2007). This activation results in the production of cytokines which eventually modulate the type of T-cell response.

Through interaction with DCs, CD₄+T-cells can differentiate into a variety of effector and regulatory subsets, including classical T-helper1 and 2 cells, regulatory T-cells (Treg)and T-helper 17 cells. It has been shown that the nature of the cytokines produced by DCs in response to various ligands determine the type of T-helper cell response.

1.2.1 Humoral Barriers to Infection

The anatomical barriers are very effective in preventing colonization of tissues by micro-organisms. However, when there is damage to tissues, the anatomical barriers are breached and infections may occur. Once infectious agents have penetrated tissues, another innate defence mechanism comes into play, namely: acute inflammation. Humoral factors play important roles in inflammation, which is characterized by oedema and the recruitment of phagocytic cells. These humoral factors are found in serum or they are formed at the site of infection.

1.2.1.1 Inflammatory Response

The inflammatory process is the reaction of blood vessels which brings about an accumulation of fluid and white blood cells in the extravascular tissues (Cotran, 1998).Infection with a pathogen triggers an acute inflammatory response in which cells and molecules ofthe immune system move into the affected site. The Activation of complement generates C3b, which coats the surface of the pathogen. Substances released from the pathogen and from damaged tissues upregulate the expression of adhesion molecules on vascular endothelium,alerting passing cells to the presence of infection. The cell-surface molecule L-selectin on neutrophils recognizes carbohydrate structures such as sialyl-Lewis X on the vascular adhesion molecules.The neutrophil rolling along the vessel wall is arrested in its course by these interactions.As the neutrophil becomes activated, it rapidly sheds L-selectin from its surface and replaces it with other cell-surface adhesion molecules, such as the integrins.These integrins bind the molecule E-selectin,which appears on the blood-vessel wall under the influence of inflammatory mediators such as bacterial lipopolysaccharide and the cytokines;interleukin-1and tumor necrosis factor-α. Complement components,prostaglandins, leukotrienes and other inflammatory mediators all contribute to the recruitment of inflammatory cells as does an important group of chemoattractant cytokines; the chemokines (Ogawa and Calhoun, 2006).

Inflammation is an important nonspecific defense reaction of tissue injury, such as that caused by a pathogen or wound (Prescott et al., 2005). It is one of the first responses of the immune system to infection (Kawai and Akira, 2006). The symptoms are redness, swelling, heat and pain, which are caused by increased blood flow into tissue. Inflammation is produced by eicosanoids and cytokines, which are released by injured or infected cells. Eicosanoids include prostaglandins that produce fever and the dilation of blood vessels associated with inflammation and leukotrienes that attract certain white blood cells (Miller, 2006). Common cytokines include interleukins that are responsible for communication between white blood cells; chemokines that promote chemotaxis and interferons that have antiviral effects, such as shutting down protein synthesis in the host cell (Leet al., 2004). Growth factors and cytotoxic factors may also be released. These cytokines and other chemicals recruit immune cells to the site of infection and promote healing of any damaged tissue following the removal of pathogens (Martins and Leibovich, 2005).Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. When tissues receive injurious stimuli, they inflame to remove them and to initiate the healing process (Ferrero-Millianiet al., 2007). It is a stereotyped response and therefore considered a mechanism of innate immunity (Abbas et al., 2007).

Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. The intravascular cells important to inflammation are neutrophils, eosinophils, basophils, lymphocytes and monocytes. The process of acute inflammation is initiated by cells already present in all tissue mainly resident macrophages, dendritic cells, mastocytes, among others. These cells contain ‘pattern recognition receptors’ (PRRs) which recognize molecules that are broadly shared by pathogens but distinguishable from host molecule and collectively referred to as pathogen-associated molecular patterns (PAMPs). At the onset of an infection, burn or other injuries; these cells undergo activation and release inflammatory mediators responsible for the clinical signs of inflammation (Cotran, 1998). Vasodilation and its resulting increased blood flow cause the redness and increased heat. Prolonged inflammation known as chronic inflammation leads to progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process (Liszewski et al., 1996). Chronic inflammation can however lead to a host of diseases such as hay fever, periodontitis, atherosclerosis, rheumatoid arthritis and even cancer.Inflammatory mediators have short half-lives and are quickly degraded in the tissue. Hence acute inflammation ceases once the stimulus has been removed.Chronic inflammation on the other hand is a more severe type of inflammation as it is prolonged.

The chemical factors produced during inflammation include histamine, bradykinin, serotonin, prostaglandins and leukotrienes. These factors sensitize pain receptors, causing vasodilation of the blood vessels at the scene and then attract phagocytes especially neutrophils(Tsvetanova et al., 1995) which then trigger other parts of the immune system by releasing factors that summon other leukocytes and lymphocytes.

1.2.3 Components of the Innate Immune System

1.2.3.1 Complement System

The complement system is a biochemical cascade that attacks the surfaces of foreign cells. It comprises an assembly of over 20 liver-manufactured, soluble and cell-bound proteins and is named for its ability to “complement” the killing of pathogens by antibodies. Complement is the major humoral components of the innate immune response (Rus et al., 2005; Mayer, 2006). Many species have complement systems, including non-mammals like fish and some invertebrates. The complement cascade helps the ability of the antibodies and phagocytic cells to clear pathogens from an organism (Janeway, 2005). It can be recruited and brought into action by the adaptive immune system. Activation of the complement cascade by protease cleavage leads to chemotaxis (C5a), inflammation and increased capillary permeability (C3a, C5a), opsonization (C3b), and cytolysis. Sequential activation of the protein components of the complement cascade upon cleavage by a protease, leads to each component’s becoming, in its turn, a protease (Rus et al., 2005). Three pathways are involved in complement attack upon pathogens: classical pathway, alternate pathway and mannose-binding lectin pathway (MBL-MAPS) (Abbas et al., 2010). The classical pathway utilizes C1, which is activated by binding of antibody to its cognate antigen. Activated C1 is a serine protease that cleaves C4 and C2 into small inactive fragments (C4a, C2a) and larger active fragments, C4b and C2b. The active component C4b binds to the sugar moieties of surface glycoproteins and binds non-covalently to C2b, forming another serine protease(Arnold et al., 2006). Macrophages and neutrophils possess receptors for C3b, so cells coated with C3b are targeted for phagocytosis (opsonization). The small C3a fragment is released into solution where it can bind to basophils and mast cells, triggering histamine release and as an anaphylatoxin, potentially participating in anaphylaxis.C3 amplifies the humoral response because of its abundance and its ability to auto-activate.The alternative pathway is not activated by antigen-antibody binding but instead relies upon spontaneous conversion of C3 to C3b, which is rapidly inactivated by its binding to inhibitory proteins and sialic acid on the cell’s surface. The lectin pathway (MBL – MASP) is homologous to the classical pathway, but utilizes opsonin, mannan-binding lectin (MBL, MBP) and ficolins rather than C1q(Liszewski et al., 1996).

1.2.3.2 Leukocytes

Leukocytes act like independent, single-celled organisms and are the second arm of the innate immune  system (Albert  et  al.,  2002). The innate leukocytes  include  the phagocytes (macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. These cells identify and eliminate pathogens, either by attacking larger pathogens Assembly of membrane through contact or by engulfing and then killing microorganisms(Janeway, 2005). Innate cells attack complex (MAC) are also important mediators in the activation of the adaptive immune system (Mayer, 2006).

Fig.1 The Complement cascade (Liszewski et al., 1996)

1.2.3.3 Phagocytes

Phagocytes generally patrol the body searching for pathogens, but can be called to specific locations by cytokines (Albert et al., 2002). Once a pathogen has been engulfed by a phagocyte, it becomes trapped in an intracellular vesicle the phagosome, which subsequently fuses with lysosome to form a phagolysosome. The pathogen is killed by the activity of digestive enzymes or following a respiratory burst that releases free radicals into the phagolysosome (Langermans et al., 1994). Phagocytosis evolved as a means of acquiring nutrients but this role was extended in phagocytes to include engulfment of pathogens as a defence mechanism. Phagocytosis probably represents the oldest form of host defence, as phagocytes have been identified in both vertebrate and invertebrate animals (Salzet et al., 2006).

1.2.3.3.1 Neutrophils, Macrophages and Dendritic Cells

These are phagocytes that travel throughout the body in pursuit of invading pathogens. Neutrophils are normally found in the bloodstream and are the most abundant type of phagocytes, normally representing 50% to 60% of the total circulating leukocytes(Stetinova et al., 1995). During the acute phase of inflammation, particularly as a result of bacterial infection, neutrophils migrate toward the site of inflammation in a process called chemotaxis and are usually the first cells to arrive at the scene of infection. Macrophages are versatile cells that reside within tissues and produce a wide array of chemicals including enzymes, complement proteins, and regulatory factors such as interleukin-1 (Bower, 2006). They serve as first line of defence during infection and help to promote immune tolerance in the steady state and also act as scavengers, ridding the body of worn-out cells and other debris (Lavin and Merad, 2013).

Dendritic cells (DC) are phagocytes in tissues that are in contact with the external environment; therefore, they are located mainly in the skin, nose, lungs, stomach, and intestines. Dendritic cells are responsible for initiating all antigen-specific immune responses. As such, they are the master regulators of the immune response and serve this function by linking the microbial sensing features of the innate immune system to the exquisite specificity of the adaptive response. They are exceptionally efficient at antigen presentation and also adept at generating just the right type of T-cells in response to a given pathogen. Importantly, DCs also help guide the immune system to respond to foreign antigens while avoiding the generation of autoimmune responses to self (Guermonprezet al., 2002; Mellman, 2013). The dendritic cell constitutes only 0.2% of white blood cell in the blood (Prescott et al., 2005).

Mast cells reside in connective tissues and mucous membranes, and regulate the inflammatory response (Krishnaswamy et al., 2006). They are most often associated with allergy and anaphylaxis. Basophils and eosinophils are related to neutrophils. They secrete chemical mediators that are involved in defending against parasites and play a role in allergic reactions, such as asthma (Kariyawasam and Robinson 2006).

1.2.3.5 Natural Killer Cells

Natural killer cells or NK cells are a component of the innate immune system which does not directly attack invading microbes. Rather, NK cells destroy compromised host cells such as tumor cells or virus-infected cells, recognizing such cells as “missing self.” This term describes cells with low levels of a cell-surface marker, the major histocompatibility complex (MHC I) – a situation that can arise in viral infections of host cells (Janeway, 2005). They were named “natural killer” because of the initial notion that they do not require activation in order to kill cells that are “missing self.” For many years it was unclear how NK cells recognize tumor cells and infected cells. It is now known that the MHC make-up on the surface of those cells is altered and the NK cells become activated through recognition of “missing self”. Normal body cells are not recognized and attacked by NK cells because they express intact self MHC antigens. Those MHC antigens are recognized by killer cell immunoglobulin receptors (KIR) which essentially put the brakes on NK cells(Janeway, 2005).

1.3 Adaptive Immune Response

The adaptive immune response consists of antibody responses and cell-mediated responses, which are carried out by different lymphocyte cells: B-cells and T-cells, respectively. Cell-mediated immunity does not involve antibodies but rather involves the activation of macrophages, natural killer cells (NK) and antigen-specific cytotoxic T-lymphocyte.

The function of adaptive immune response is to destroy invading pathogens and any toxic molecules they produce. The ability to distinguish what is foreign from what is self is a fundamental feature of the adaptive immune system.

Allergic conditions such as hayfever and asthma are examples of deleterious adaptive immune responses against apparently harmless foreign molecules (Pancer and Cooper, 2006). The major function of the acquired immune system includes the recognition of specific non-self antigen in the presence of self during the antigen presentation process, the generation of responses that are tailored to maximally eliminate specific pathogens or pathogen-infected cells and development of immunological memory, in which each pathogen is remembered by a signature antibody or T-cell receptors (Prescott et al., 2005).

1.3.1 The Lymphocytes

Lymphocytes are kinds of white blood cells in the vertebrates’ immune system that are specifically the landmark of the adaptive immune system. It can be divided into large lymphocytes and small lymphocytes.Large granular lymphocytes include natural killer cells (NK cells) while small lymphocytes consist of T-cells and B-cells.B-cells are involved in the humoral immune response mediated by secreted antibodies, whereas T-cells are involved in the activation of phagocytes, natural killer cells and antigen-specific cytotoxic T-lymphocytes (Janeway et al., 2001)

1.3.2 Helper T-Cells

Helper T-cells are arguably the most important cells in adaptive immunity as they are required for almost all adaptive immune responses. They do not only activate B-cells to secrete antibodies and macrophages to destroy ingested microbes, but they also help activate cytotoxic T-cells to kill infected target cells. Helper T-cells regulate both innate and adaptive immune responses and help determine which immune response the body makes to a particular pathogen (McHeyzer-Williams et al., 2006). As dramatically demonstrated in AIDS, without helper T-cells the body becomes defenceless even against many microbes that are normally harmless.Helper T-cells themselves however can only function when activated to become effector cells. They are activated on the surface of antigen-presenting cells which mature during the innate immune responses triggered by an infection. The innate responses also dictate what kind of effector cell a helper T-cell will develop into and thereby determine the nature of the adaptive immune response elicited.To activate a cytotoxic or helper T-cell to proliferate and differentiate into an effector cell, an antigen-presenting cell provides two kinds of signals.

Signal 1 is provided by a foreign peptide bound to a major histocompatibility complex (MHC)protein on the surface of the presenting cell. This peptide-MHC complex signals through the T-cell receptor and its associated proteins. Signal 2 is provided by co-stimulatory proteins, especially the B7 proteins, which are recognized by the co-receptor protein CD28 on the surface of the T-cell. The expression of B7 proteins on an antigen-presenting cell is induced by pathogens during the innate response to an infection. Effector T-cells act back to promote the expression of B7 proteins on antigen-presenting cells, creating a positive feedback loop that amplifies the T-cell response. When an antigen-presenting cell activates a naïve helper T-cell in a peripheral lymphoid tissue, the T-cell can differentiate into either a T_H1 or T_H2 effector helper cell. These two types of functionally distinct subclasses of effector helper T-cells can be distinguished by the cytokines they secrete. If the cell differentiates into a T_H1 cell, it will secrete interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) and will activate macrophages to kill microbes located within the macrophages’ phagosomes. It will also activate cytotoxic T-cells to kill infected cells. In these ways, T_H1 cells mainly defend an animal against intracellular pathogens. They may also stimulate B-cells to secrete specific subclasses of IgG antibodies that can coat extracellular microbes and activate complement.If the naïve T-helper cell differentiates into a T_H2 cell, by contrast, it will secrete interleukins 4, 5, 10, and 13 (IL-4, IL-5, IL-10, and IL-13) among others and will mainly defend the animal against extracellular pathogens. A T_H2 cell can stimulate B-cells to make most classes of antibodies, including IgE and some subclasses of IgG antibodies that bind to mast cells, basophils and eosinophils. These cells release local mediators that cause sneezing, coughing or diarrhoea and help expel extracellular microbes and larger parasites from epithelial surfaces of the body.Thus the decision of naïve helper T-cells to differentiate into T_H1 or T_H2 effector cells influences the type of adaptive immune response that will be mounted against the pathogen. The specific cytokines present during the process of helper T-cell activation influence the type of effector cell produced. Many parasitic protozoa and worms stimulate the production of cytokines that encourage T_H2 development and thereby antibody production and eosinophil activation leading to parasite expulsion.

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