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.






Title Page





Table of Contents

List of Figures

List of Tables

List of Abbreviations



1.1 Introduction

1.1.1 Immuno-modulation Immuno-stimulation Immuno-suppression

1.2 Innate immune system

1.2.1 Humoral barriers to infection Inflammatory response

1.2.2 Components of the innate immune system Complement system Leukocytes Phagocytes Neutrophils, Macrophages and Dendritic cells 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 Basic immunoglobulin structure Immunoglobulin production Classes or isotypes of immunoglobulin Immunoglobulin M Immunoglobulin G Immunoglobulin D Immunoglobulin A Immunoglobulin E

1.4 Cytokines

1.4.1 Class of cytokines Chemokines Interferons Interferons-γ Interleukins Interleukin-2 Interleukin-10 Tumor necrosis factor Tumor necrosis factor-alpha (TNF-α)

1.5 Pyrogallol

1.6 Antioxidants

1.6.1 Types of antioxidants

1.6.2 Classifications of antioxidants Functions of antioxidants Glutathione

1.6.3 Tocopherols and tocotrienols (vitamin E)

1.6.2    Antioxidant enzymes

1.6.3    Superoxide dismutase 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



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 Aqueous extract Methanol extract 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 Moisture content Crude fibre Total ash Crude fat Crude protein Carbohydrate

2.2.5    Qualitative phytochemical analysis of leaves of Telfairia occidentalis and Tectona grandis Test for alkaloids Test for flavonoids Test for glycosides Test for saponins Test for tannins Test for terpenoids and steroids

2.2.6    Quantitative phytochemical analysis of T. occidentalis and T. grandis Alkaloid determination Flavonoids determination Steroids determination Terpenoid Tannin Glycosides Cyanogenic glycosides Soluble carbohydrates Reducing sugars

2.2.7    Animals Antigen

2.2.8 Experimental design First stage Second stage Third stage Final stage

2.2.9    Preliminary screening of ethanol and aqueous extracts for immunomodulatory activity Studies on delayed type hypersensitivity response (DTHR) Studies on humoral antibody (HA) response

2.2.10  Haematological assay Determination of erythrocyte count by haemocytometry Determination of total leucocyte count by haemocytometry Packed Cell Volume (PCV) estimation Determination of Haemoglobin (Hb) concentration Determination of CD4+ count

2.2.11Determination of enzymatic antioxidants Estimation of superoxide dismutase Estimation of catalase Estimation of glutathione peroxidise

2.2.12 Non-enzymatic antioxidants Estimation of reduced glutathione Determination of selenium Estimation of vitamin E (alpha tocopherol)

2.2.13  Estimation of extent of lipid peroxidation (malondialdehyde) Serum calcium determination Serum zinc determination Serum iron determination

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



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


4.1       Discussion

4.2       Conclusion

4.3       Contribution to Knowledge

4.4       Recommendations for further research





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



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






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








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 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. Immuno-stimulatory 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 immune-modulants 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 immune-modulatory 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 seriousinvestigation 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). Thereis 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; Vinothapooshan and Sundar, 2011). Immuno-modulatorsalter the immune system by interacting with it to up-regulate or down-regulate specific aspect of the host response (Stanilove 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 immunoregulator 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 antigenomimetics 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:immuno-stimulation and immuno-suppression. Immuno-stimulation


Immuno-stimulation is a process that involves the activation of the immune system activity and that of its components. Immuno-stimulatory agents are used often to achieve this purpose and are grouped into specific and non-specific 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). Immuno-suppression


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, immuno-suppression can be induced in some special cases such asin organ transplantto prevent graft rejection and in treating auto-immune 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 effectorand regulatory subsets, including classical T-helper1 and 2 cells, regulatory T-cells (Treg)and T-helper17 cells. It has been shown that the nature of the cytokines produced by DCs inresponse 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. Inflammatory Response


The inflammatory process is the reaction of blood vessels which brings about an accumulation of fluid and white blood cells in the extra vascular tissues (Cotran, 1998).Infection with a pathogen triggers an acute inflammatoryresponse in which cellsand molecules ofthe immune system move into the affected site. Theactivation ofcomplement generates C3b, which coatsthe surface of the pathogen. Substancesreleased from the pathogen and from damaged tissuesup-regulate the expression of adhesion moleculeson vascular endothelium,alerting passing cells tothe presence of infection. The cell-surface moleculeL-selectin on neutrophils recognizes carbohydratestructures such as sialyl-LewisXon the vascular adhesionmolecules.The neutrophil rolling along thevesselwall is arrested in its course by these interactions.As the neutrophil becomes activated, it rapidlysheds L-selectin from its surface and replaces it withother cell-surface adhesion molecules, such as the integrins.These integrins bind the molecule E-selectin,which appears on the blood-vessel wall under the influenceof inflammatory mediators such as bacteriallipopolysaccharide and the cytokines;interleukin-1and tumor necrosis factor-α. Complement components,prostaglandins, leukotrienes and other inflammatorymediators all contribute to the recruitmentof inflammatory cells as does an important group ofchemoattractant cytokines; the chemokines (Ogawa and Calhoun, 2006).


Inflammation is an important non-specific 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 anti-viral 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 aninfection, 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(Stvitnovaet 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 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. Activationof the complement cascade by protease cleavage leads to chemotaxis (C5a), inflammation and increased capillary permeability (C3a, C5a), opsonization (C3b), and cytolysis. Sequential activationof the protein components of the complement cascadeupon 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, C4band 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 targetted 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 toinhibitory proteins and sialic acidon the cell's surface. The lectin pathway (MBL - MASP) is homologous to the classical pathway, but utilizes opsonin, mannan-binding lectin (MBL, MBP) and ficolinsrather than C1q(Liszewski et al., 1996).





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 croorganisms Celllysis (Janeway, 2005). Innate cells attach complex (MAC) are also important mediators in the activation of the adaptive immune system (Mayer, 2006). 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 (Salzetet al., 2006)., 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(Stvrtinovaet 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). 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).



1.3Adaptive 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 (Prescottet 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 bysecreted antibodies, whereas T-cells are involved in the activation of phagocytes, natural killer cells and antigen-specific cytotoxic T-lymphocytes (Janewayet 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 amajor 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 TH1 or TH2 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 TH1 cell, it willsecrete 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, TH1 cells mainly defend an animal against intracellular pathogens. They may also stimulate B-cells to secrete specific sub-classes of IgG antibodies that can coat extracellular microbes and activate complement.If the naïve T-helper cell differentiates into a TH2 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 TH2 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 TH1 or TH2 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 wormsstimulate the production of cytokines that encourage TH2 development and thereby antibody production and eosinophil activation leading to parasite expulsion.


1.3.3 Killer T-Cells








Killer T-cells destroy target cells only when specifically activated by helper T-cells. There also exist the NKT-cells (Natural Killer Cells) that are heterogeneous group of T-cells sharing both the properties of T-cells and natural killer (NK) cells. Generally, the NKT cells refer to CD-id


restricted AntigenT-cellsprespresentinghumans and mice, some of which co-express a nearly biased, semi-invariant T-cell receptor(TCR) and NK cells marker (Godfrey et al., 2004). NKT cells differ from NK cells as they recognize non-polymorphic CD-id molecule, an antigen presenting molecule that binds self, foreign lipids and glycolipids, hence constituting approximately 0.1% of all peripheral blood T-cells (Jerudet al., 2006).



1.3.4 The B-Cells


The B-cells work chiefly by secreting antibodies into the body’s fluid. B-cells identify pathogens when antibodies on its surface bind to a specific foreign antigen (Sproulet al., 2000). Principal functions of B-cells are to make antibodies against antigens, to perform the role of antigen-presenting cells (APCs)and to develop into memory B-cells after activation of antigen interaction (Mauriceet al., 2008). In mammals, immature B-cells are formed in the bone marrow, which is used as a backbone for the cells’ name (Albert et al., 2002). When the B-cell fails in any step of the maturation process, it will die by apoptotic mechanism known as clonal deletion (Parham,2005). When the B-cells on the surface of the cell matches the detected antigens present in the body, it proliferates and secretes a free form of those receptors (antibodies) with identical binding sites as the ones on the original cell surface. Memory B-cells are equally formed to recognize the same antigen. Thus inflammation would then be used as a part of the adaptive immune system for a more efficient and more powerful immune response in a future encounter with that antigen. and Function of Immunoglobulins


Immunoglobulins are heterodimeric proteins composed of two heavy (H) and two light (L) chains. They can be separated functionally into variable (V) domains that bind antigens and constant (C) domains that specify effector functions such as activation of complement or binding to Fc receptors (Williams and Barclay,1988). Immunoglobulins generally assume one of two roles: they may act as plasma membrane bound antigen receptors on the surface of a B-cell or as antibodies free in cellular fluids functioning to intercept and eliminate antigenic determinants. In either role, antibody function is intimately related to its structure (Harpaz and Chothia, 1994). Basic Immunoglobulin Structure


Immunoglobulins are composed of four polypeptide chains: two "light" chains (lambda or kappa) and two "heavy" chains (alpha, delta, gamma, epsilon or mu). The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, IgM respectively). Light chains are composed of 220 amino acid residues while heavy chains are composed of 440-550 amino acid residues. Each chain has "constant" and "variable" regions. Variable regions are contained within the amino (NH2) terminal end of the polypeptide chain (amino acids 1-110). "Hypervariable" regions, or "Complementarity Determining Regions" (CDRs) are found within the variable regions of both the heavy and light chains. These regions serve to recognize and bind specifically to antigen. The four polypeptide chains are held together by disulfide (-S-S-) bonds (Torres et al., 2008). Immunoglobulin Production


The production of immunoglobulins by B-cells or plasma cells occurs in different stages. During differentiation of B-cells from precursor stem cells, rearrangement, recombination and mutation of the immunoglobulin V, D, and J regions occur to produce functional VJ (light chain) and VDJ (heavy chain) genes(Dudley et al., 2005). Initially a mature B-cell will produce primarily IgD (and some membrane IgM) that will migrate to the cell surface to act as the antigen receptor. Upon stimulation by antigen, the B-cell will differentiate into a plasma cell expressing large amounts of secreted IgM. Some cells will undergo a "class switch" during which a rearrangement of the DNA will occur, placing the VDJ gene next to the genes encoding the IgG, IgE or IgA constant regions. Upon secondary induction (i.e. the secondary response), these B-cells will differentiate into plasma cells expressing the new isotype. Most commonly, this results in a switch from IgM (primary response) to IgG (secondary response). The factors that lead to production of IgE or IgA instead of IgG are not well understood (Vincke et al., 2009) or Isotypes of Immunoglobulin Immunoglobulin M (IgM)


IgM is the first immunoglobulin expressed during B-cell development. Naïve B cells express monomeric IgM on their surfaces and associate with CD79a and CD79b polypeptide chains that participate in IgM cell signaling.IgM functions by opsonizing (coating) antigen for destruction and fixing complement. The pentameric nature of the antibody renders it very efficient in this process.IgM antibodies are associated with a primary immune response and are frequently used to diagnose acute exposure to an immunogen or pathogen. They also play a role in immunoregulation (Nimmerjahn and Ravtech, 2006). G (IgG)


IgG is the predominant isotype found in the body. It has the longest serum half-life of all immunoglobulin isotypes. Based on structural, antigenic and functional differences in the constant region of the heavy chain, four IgG subclasses (IgG1, IgG2, IgG3 and IgG4) have been identified. The IgG sub-classes exhibit different functional activities. Activation of the complement cascade is an important means of clearance of opsonized pathogens (Siberilet al., 2006). There are also similarities within the sub-classes such as transplacental transport and participation in the secondary immune response. Within the secondary antibody response, there is skewing in the predominant subclass that is induced. For example, IgG1 and IgG3 antibodies are generally induced in response to protein antigens whereas IgG2 and IgG4 are associated with polysaccharide antigens. Specific subclasses may be associated with individual disease processes. For example, in pemphigus vulgaris, a mucocutaneous blistering disease, IgG4 antibodies to desmoglein 3 are pathogenic (Yeh et al., 2006) whereas first degree relatives with IgG1 autoantibodies to the same protein show no evidence of the disease. In HIV, it has been shown that IgG3 antibodies may be more effective at neutralizing virus than IgG1 antibodies, presumably through an increase in antibody flexibility improving antibody access or inducing changes in the oligomer structure of the virus (Cavacini et al., 2003). Immunoglobulin D (IgD)


Circulating IgD is found at very low levels in the serum with a short serum half-life which may be attributed to sensitivity of the molecule with the hinge region in particular to proteolysis. Circulating IgD can react with specific bacterial proteins, such as the IgD binding protein of Moraxella catarrhalis independent of the variable regions of the antibody (Riesbeck and Nordstrom, 2006). The binding of these bacterial proteins to the constant region of IgD results in B-cell stimulation and activation. IgD is expressed on the membranes of B-cells when they leave the bone marrow and populate secondary lymphoid organs (Geisberger et al., 2006). A (IgA)


IgA serum levels tend to be higher than IgM but considerably lower than IgG. Conversely, IgA levels are much higher than IgG at mucosal surfaces and in secretions, including the saliva and breast milk (Woof and Mestecky, 2005). In particular, IgA can contribute up to 50% of the protein in colostrum. There are two subclasses of IgA; IgA1 and IgA2 whose structures differ mainly in their hinge regions. IgA1 has a longer hinge region with a duplicated stretch of amino acids that is lacking in IgA2. This elongated hinge region increases the sensitivity of IgA1 to bacterial proteases in spite of partial protection by glycans. Such increased protection against protease digestion may explain why IgA2 predominates in the many mucosal secretions, such as the genital tract, whereas more than 90% of serum IgA is in the form of IgA1(Corthesy, 2007).IgA is critical at protecting mucosal surfaces from toxins, viruses and bacteria by direct neutralization or by prevention of binding to the mucosal surface. Intracellular IgA may also be important in preventing bacterial or viral infection and/or pathogenesis. It has been proposed that IgA may also act as a potentiator of the immune response in intestinal tissue by uptake of antigen to dendritic cells (Corthesy, 2007). Immunoglobulin E (IgE)


IgE is a very potent immunoglobulin, though present at the lowest serum concentration and with the shortest half-life. It is associated with hypersensitivity and allergic reactions as well as the response to parasitic worm infections. IgE binds with extremely high affinity to the FcεRI which is expressed on mast cells, basophils, langerhans cells and eosinophils. Circulating IgE up-regulates FcεRI expression on these cells. The combination of strong binding and up-regulation of FcεR expression contributes to the remarkable potency of this immunoglobulin(Chang et al., 2007).





Cytokines (Greek cyto, cell and kinesis movement) is a generic term for any soluble proteins or glycoproteins released by one cell population that acts as an intercellular (between cells) mediator or signaling molecule (Joanne et al., 2011). They are multifunctional proteins whose biological properties suggest a key role in haemopoiesis, immunity, infectious disease, tumorigenesis, homeostasis, tissue repair, cellular development and growth (Maurice et al., 2008). Cytokines, a large group of soluble extracellular proteins or glycoproteins, are key intercellular regulators and mobilizers(Kelvin et al., 2006). When released from mononuclear phagocytes, these proteins are called monokines; when released from T-lymphocytes, they are called lymphokines; when produced by a leukocytes and the action is on another leukocyte, they are called interleukines; and if their effect is to stimulate the growth and differentiation of immature leukocytes in the bone marrow, they are called colony stimulating factors. Cytokines have been shown to be crucial to innate and adaptive inflammatory responses, cell growth and differentiation, cell death, angiogenesis and developmental as well as repair processes (Oppenheim, 2001). In addition, cytokines provide links between organsand systems, providing molecular cues for maintaining physiological stability(O’sullivan et al.,1998). They usually act as signaling molecules by binding to their own glycoprotein receptors on cell membranes. This initial interaction is followed by a relay of the signal to the cell nucleus. Signal transduction is mediated as in many hormone-receptor systems by kinase-mediated phosphorylation of cytoplasmic proteins.


Cytokines operate both as a cascade and as a network, regulating the production of other cytokines and cytokine receptors, while stimulating the production of acute-phase proteins (Gabay and Kushner, 1999). As models of physiology continue to develop beyond compartmentalized organ systems, elucidation of the global activity of cytokines offers further support to an expanding understanding of cell-to-cell communication. The inflammatory processes of cardiovascular disease are one such example. Beyond leukocytes, the liver, heart, vessel walls, and adipose tissue are known to produce cytokines; thus any of these tissues may potentially contribute to the inflammatory nature of cardiovascular disease (Rader, 2000). Modulation of cytokine secretion may offer novel approaches in the treatment and defense of a variety of diseases. One strategy in the modulation of cytokine expression may be through the use of herbal medicines (Kelvin et al., 2006). Because of their major participatory role in nearly all pathophysiologic processes and their therapeutic potential, there is a need to identify and measure cytokines. In the clinical laboratory, cytokine assessment has been used to monitor disease progression and activity. As a consequence of these expanding applications, the future use of cytokine monitoring in the clinical and research setting will undoubtedly increase (Remick, 2006).


As a result of the growing recognition of cytokine activities, altering cytokine expression and targeting their receptors may offer therapeutic potential. Pharmacological strategies include cytokine antagonist, agonist, inhibitors, and stimulators (Sommer, 1999; Luster et al., 2005). Novel approach now used in the treatment of asthma is the inhibition of T helper 2 (TH2)-derived cytokine expression, resulting in downstream effects on IgE and eosinophils (Vileck and Feldmann 2004). Interleukin-10 (IL-10) demonstrates modulation of brain inflammation, which may have application for conditions such asAlzheimer’s disease. In addition, interleukin-2 (IL-2) and interleukin-12 (IL-12) in combination may provide a potential therapeutic approach for neuroblastomas (Abbas et al., 1994).



1.4.1 Classesof Cytokines


Cytokines has been grouped into the following categories or families: Chemokines, Interleukines(IL), Interferons (IFN) and Tumor Necrosis Factor(TNF) (Joanne et al., 2011).Based on the structural homologies of their receptors, these proteins were initially believed to act primarily as anti-neoplastic agents, but are now seen to be crucial to innate and adaptive immune response, cell growth and differentiation, cell death and developmental as well as repair processes(Young and Cummins,1990; Oppenhein, 2001; Terlikowski, 2001). Chemokines


This class of cytokine, stimulates chemotaxis and chemokinesis (they direct cell movement) and thus play an important role in the acute inflammatory responses (Joanneet al., 2011).Chemokines are a superfamily of small proteins with a crucial role in immune and inflammatory reactions. Induction of leukocyte migration is the essential function of chemokines and their specific receptors but they also affect angiogenesis, collagen production, and proliferation of haemopoietic precursors (Medoff and Luster, 2006). Interferons


Interferons (IFN) are proteins made and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites or tumor cells. They allow for communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors (Wikipedia, 2013).Interferons are named after their ability to "interfere" with viral replication within host cells. In addition IFNs activate immune cells, such as natural killer cells and macrophages, they increase recognition of infection or tumor cells by up-regulating antigen presentation to T-lymphocytes and they increase the ability of uninfected host cells to resist new infection by virus. Certain symptoms, such as aching muscles and fever are related to the production of IFNs during infection (Prestkaet al., 2004).About ten distinct IFNs have been identified in mammals; seven of these have been described for humans. They are typically divided among three IFN classes based on the type of receptor through which they signal: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all IFN classes are very important for fighting viral infection.The type I interferon present in humans are IFN-α, IFN-βand IFN-ω. In humans IFN-γ is the only type II interferon.Acceptance of interferon type III is less universal than that of type I and type II (De-Weerdet al., 2007). The different interferons have overlapping biological activities such as antiviral actions, anti-proliferative and immune-regulatory actions. However non-overlapping functions also exist. For example, IFN-β is used to successfully treat patients with multiple sclerosis (MS)whereas IFN-γ has been shown to exacerbate the disease. Interferon (IFN-γ)


This is a dimerized soluble cytokine that is the only member of the type II class of interferons. IFN-γ is a cytokine that is critical for innate and adaptive immunity against viral and intracellular bacterial infections and for tumor control. It is an important activator of macrophages (Nagineni et al., 2007). Aberrant IFN-γ expression is associated with a number of autoinflammatory and autoimmune diseases (Banchereau and Paschal, 2006). The importance of IFN-γ in the immune system stems in part from its ability to inhibit viral replication directly and most importantly from its immunomodulatory effects. IFN-γ is produced predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response and binds CD4 Th1 and CD8 cytotoxic T-lymphocyte (CTL) effector T-cells once antigen-specific immunity develops (Sadiret al., 1998;Serio et al., 2014).IFN-γ has anti-viral, immunoregulatory, and anti-tumor properties. It alters transcription in up to 30 genes producing a variety of physiological and cellular responses. Among the effects are:


·        Promotion of NK cell activity

· Increasing antigen presentation and lysosome activity of macrophages(Schroederet al.,2004). Interleukins


Interleukins are a group of cytokines that were first seen to be expressed by white blood cells (leukocytes). The function of the immune system depends in a large part on interleukins and rare deficiencies of a number of them have been described all featuring autoimmune diseases or immune deficiency. The majority of interleukins are synthesized by helper CD4 T-lymphocytes, as well as through monocytes, macrophages and endothelial cells. They promote the development and differentiation of T and B-lymphocytes and haemopoietic cells (Brocker et al.,2010). Interleukins not only initiate communication among immune cells, but they can also induce profound effects on non-immune cells. 2 (IL-2)


Interleukin 2 (IL-2) is a protein that regulates the activities of white blood cells (leukocytes,often lymphocytes) that are responsible for immunity and also a hormone-like mediator of the immune system (Smith et al.,1980).The first function attributed to IL-2 was a potent capacity to enhance in vitro T-cell proliferation and differentiation (Gillis et al., 1978) and it was therefore originally named T-cell growth factor (TCGF). Interleukin-2 was also assumed to have a crucial rolein vivo during antigen-driven clonal expansion of T-cells (Waldmann, 2006). As IL-2 is mainly produced by activated T-cells and in particular by activatedCD4+ T-helper cells, at least part of their ‘helper’ fu nction for CD8+ T-cells was attributed to IL-2.The IL-2/IL-2R interaction stimulates the growth, differentiation and survival of antigen-specific CD4+ T-cells and CD8+ T-cells. Subsequentto these initial descriptions of the functions of IL-2, numerous studieshave highlighted many more seemingly contradictory functions of this cytokine with respect to immune-enhancing functions.IL2 has a role in supporting proliferation (Gillis and Smith 1977) and survival. There is evidence that IL-2 is an important factor that allows the generation of memory T-cells, which are able to undergo secondary expansion when they re-encounter an antigen. IL-2 may exhibit anti-inflammatory properties at some instances, as do other pro-inflammatorycytokinessuch as interferon-γ (Bachmann and Kopf 2002; Laurence, 2007). Interleukin-10


Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T-cells, monocytes and macrophages, is a multifunctional cytokine with diverse effects on most haemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B-cells, NK cells, cytotoxic and helper T-cells, mast cells, granulocytes, dendritic cells, keratinocytes and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T-cell, the T-regulatory cell, which may feature prominently in control of immune responses and tolerance in vivo. Uniquely among haemopoietic cytokines, IL-10 has closely related homologous genes in several virus genomes, which testify to its crucial role in regulating immune and inflammatory responses. Anti-inflammatory mediators, especially IL-10 has been shown to play greater roles in counterbalancing the pro-inflammatory response in various infectious diseases (Sharmaet al., 2004). Tumour Necrosis Factor(TNF)


Tumor necrosis factor (TNF) refers to a group of cytokines that can cause cell death (apoptosis). The first two members of this family to be identified were tumor necrosis factor-alpha (TNF-α) and tumor necrosis factor-beta (TNF-β). Tumor Necrosis Factor-Alpha (TNF-α)


TNF-α is a multifunctional cytokine involved in many different pathways, in homeostasis and pathophysiology of mammals. It is essentially a pro-inflammatory cytokine. However it can show opposing biological effects suggesting complex regulatory mechanisms. TNF-α, also known as cachectin was first detected as a cytotoxic factor inducing lysis of certain tumor cells. The TNF-α gene is a member of the TNF-super family consisting of at least 20 distinct members that act through their receptors. TNF-α release is mainly triggered by viral infections and endotoxins, lipopolysaccharides or other bacterial components, by tissue injury, DNA-damage and by IL-1 and TNF-α itself. It is primarily expressed in macrophages, but also in monocytes, neutrophils, NK-cells, mast-cells, endothelial cells and activated lymphocytes. The expression of other cytokines, chemokines, reactive oxygen intermediates, nitric oxide and prostaglandins is stimulated by TNF-α (Wajantet al., 2003). It promotes local or systemic inflammation and stimulates the acute phase response. Very high expressions of TNF-α after infection can lead to septic shock whereas sustained low levels induce cachexia and inflammation (Serio et al.,


2014)Tumor necrosis factor-αsecretion is one of the major effector mechanisms of memory CD8+ T-cells believed to be required for immunologicalprotection in vivo. Inflammatory cytokines play an important role in controlling infections. TNF-α is therefore an essential cytokine in the control of inflammatory lesions (Allenbach et al., 2008). TNF-α isalsoconsidered as an important inflammatory mediator for cell recruitment and healingof pathogen-induced lesions (Matte and Olivier 2002). However excessive production of TNF-αcontributes to disease manifestation by damaging neighbouring cells as demonstrated by various investigators (Craig et al., 1989; Skworet al., 2008).





Pyrogallol (1, 2, 3-trihydroxybenzene) is a white crystalline soluble solid. It is a powerful reducing agent usually present in an alkaline solution and rapidly absorbs oxygen (Khan and Kahn 2006). Pyrogallol is an immune suppressing chemical and it induces humoral as well as cell-mediated immune-suppression (Chanduaet al., 2011). Pyrogallol was employed to suppress the immunity of the experimental rats. In analytical chemistry, pyrogallol is used as a complexing agent, reducing agent and in alkaline solution as an indicator of gaseous oxygen (Mercado et al., 2013).

Fig. 3: Structure of pyrogallol (C6H6O3)



1.6    Antioxidants


Antioxidants are compounds that delay the start or slow the rate of lipid oxidation reaction. They inhibit the formation of free radicals and hence contribute to the stabilization of the lipid samples. They play an important role in the body due to favorable effects on human health.


Consumption of foods containing phytochemicals with potential antioxidantproperties can reduce the risk of human disease (Temple,2000).Some antioxidants are only found in a few organisms (Miller and Britigan, 1997). The relative importance and interactions between these different antioxidants is a very complex question, with the various metabolites and enzyme systems having synergistic and interdependent effects on one another (Sies, 1997; Chaudière and Ferrari-Iliou,1999). The action of one antioxidant may therefore depend on the proper functioning of other members of the antioxidant system. The amount of protection provided by any one antioxidant will also depend on its concentration, its reactivity towards the particular reactive oxygen species being considered and the status of the antioxidants with which it interacts (Vertuani et al., 2004). Some compounds contribute to antioxidant defense by chelating transition metals and preventing them from catalyzing the production of free radicals in the cell. Particularly important is the ability to sequester iron, which is the function of iron-binding proteins such as transferrin and ferritin (Imlay, 2003). Selenium and zinc are commonly referred to as antioxidant nutrients. They have no antioxidant action themselves and are instead required for the activity of some antioxidant enzymes.


Naturally, there is a dynamic balance betweenthe amount of free-radicals generated in the body andantioxidants to quench and or scavenge them and protectthe body against their deleterious effects. However the amounts of these protective antioxidantprinciples present under the normal physiologicalconditions are sufficient only to cope with thephysiological rate of free-radical generation (Ashok andSushil 2005).



1.6.1 Types of Antioxidants


1.    Primary or chain breaking antioxidants (scavenger antioxidants): These antioxidants can neutralize free radicals by donating one of their own electrons, ending the electron “stealing” reaction.


2.  Secondary or preventive antioxidants: They act through numerous possible mechanisms like a) Sequestration of transition metal ions which are not allowed to participate in metal catalyzed reactions.


b) Removal of peroxides that can react with transition metal ions to produce reactive oxygen species(ROS).


c) Removal of reactive oxygen species (ROS) etc.


3.   Tertiary antioxidant defences: These are the repair processes which remove damaged bio-molecules before they can accumulate and their presence results in altered cell metabolism and viability e.g. damaged DNA repaired by enzyme methionine sulphoxide reductase (Cheeseman and Slater,1993).



1.6.2 Classificationof Antioxidants:


Antioxidants are classified into two broad divisions depending on whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). In general water-soluble antioxidants react with oxidants in the cell cytosol and the blood plasma, while lipid-soluble antioxidants protect cell membranes from lipid peroxidation. These compounds may be synthesized in the body or obtained from the diet (Vertuaniet al., 2004). Chain breaking antioxidants are highly reactive with free radicals and form stable compounds that do not contribute to the oxidation chain reaction.


Antioxidants are believed to play very important role in the body defense system against reactive oxygen species (Boxin et al., 2002; Viveck and Surrendra 2006). Natural antioxidants are constituents of many fruits and vegetables and they have attracted a great deal of public and scientific attention. Dietary antioxidants such as ascorbates, tocopherols and carotenoids are well known (Boskou, 2006). The different antioxidants are present at a wide range of concentrations in body fluids and tissues, with some such as glutathione or ubiquinone mostly present within cells, while others such as uric acid are more evenly distributed.



1.6.2 Functions of Antioxidants


In the body, all antioxidants are working in concert as a team responsible for prevention of the damaging effects of free radicals and toxic products of their metabolism.Four possible mechanisms have been suggested by which antioxidants function to reduce the rate of oxidation of fats and oils. These are:


·        Hydrogen donation by the antioxidant.


·        Electron donation by the antioxidant.


·        Addition of lipid to the antioxidant.


·        Formation of a complex between the lipid and antioxidant.


However, it is thought that the first two mechanisms are the most probable modes of action of antioxidants.





Glutathione is a cysteine-containing tripeptide found in most forms of aerobic life. It is not required in the diet and is instead synthesized in cells from its constituent amino acids. Glutathione has antioxidant properties since the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, glutathione is maintained in the reduced form by the enzyme glutathione reductase and in turn reduces other metabolites and enzyme systems such as ascorbate in the glutathione-ascorbate cycle, glutathione peroxidases and glutaredoxins as well as reacting directly with oxidants. Due to its high concentration and its central role in maintaining the cell's redox state, glutathione is one of the most important cellular antioxidants (Fahey, 2001; Sharma et al., 2004).



1.6.4 Tocopherols and Tocotrienols (Vitamin E)


Vitamin E is the collective name for a set of eight related tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties (Herrera and Barbas, 2001; Packer et al.,


2001). Of these, α-tocopherol has been most studied as it has the highest bioavailability with the body preferentially absorbing and metabolizing this form. It has been claimed that the α-tocopherol form is the most important lipid-soluble antioxidant and that it protects membranes from oxidation by reacting with lipid radicals produced in lipid peroxidation chain reaction (Herrera and Barbas, 2001; Traber and Atkinson, 2007). This removes the free radical intermediates and prevents the propagation reaction from continuing. This reaction produces oxidized α-tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol (Wang et al., 2004). This is in line with findings showing that α-tocopherol, but not water-soluble antioxidants, efficiently protects glutathione peroxidase 4 (GPX4)-deficient cells from cell death (Seiler et al., 2008). GPx4 is the only known enzyme that efficiently reduces lipid-hydroperoxides within biological membranes. In general, antioxidants are categorized into:


Antioxidant enzymes like superoxide dismutase, catalase and gluthathione peroxidase. Non-enzyme antioxidants which include:


Minerals e.g. zinc, selenium, glutathione. Vitamins e.g. vitamin A, vitamin C and vitamin E.


Carotenoids e.g. lycopene, β-carotene, lutein, zeaxanthin. Flavonoids e.g. xanthones, flavonols and flavones.


1.6.5 Antioxidant Enzymes


Life in oxygen has led to the evolution ofbiochemical adaptationsthat exploit the reactivity of reactive oxygen species (ROS). The term ROS is generic,embracing free radicals such as superoxide,hydroxyl radicals and singlet oxygen (Graham andChristine, 1998). The antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase/reductase, convert reactive oxygen species into non-reactive oxygen molecules (Le et al., 2004). Antioxidant enzymes are an important protective mechanism against ROS and their effectiveness vary with the stage of development and other physiological aspects of the organism. The most important antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) (Jinghua et al., 2007). Dismutase


Superoxide dismutase is an endogenous and first-line-of-defense enzyme that eliminates superoxide by catalyzing its dismutation into oxygen and hydrogen peroxide(Okado-Matsumoto and Fridovich,2001; Henry et al., 2006; Vivek and Surendra, 2006).


Superoxide radical (O2-) isgenerated as by-product inaerobic organisms from anumber of physiological reactions and redox reactions incells (Andrea et al., 1989). It can react with hydrogenperoxide (H2O2) to produce hydroxyl radical (-OH) oneof the most reactive molecules in the living cells.Hydroxyl radicals can cause the peroxidation of membranelipids. To ameliorate the damage caused by hydroxylradicals, superoxide radicals and hydrogen peroxide,organisms have evolved mechanisms to control theconcentration of these reactants (Campana et al., 2004).Superoxide dismutase is animportant enzyme family in living cells for maintainingnormal physiological conditions and for coping withstress (Olawale et al., 2008).


Catalases are the class of enzymes, which catalyze the decomposition of hydrogen peroxide to oxygen and water and these ubiquitous enzymes have been isolated and purified from different natural sources including animal tissues, plants and micro-organisms (Malcolm et al., 1977). Catalase converts H2O2to O2and H2Oin concert with the tripeptide glutathione.


2H2O2           2H2O + 2O2

Some of the pathways for the generation of hydrogen peroxide include the following:


a)superoxide dismutase which promote the disproportionation of superoxide into oxygen and hydrogen peroxide.


b)Degradation of adenosine monophosphate which yields hypoxanthine. Hypoxanthine is then oxidatively catabolized first to xanthine and then to uric acid, and the reaction is catalyzed by the enzyme xanthine oxidase (Kirk and Ronald, 1988).


Degradation of hypoxanthine to uric acid to form hydrogen peroxide(XO = xanthine oxidase).


c)Degradation of guanosine monophosphate yields xanthine as an intermediate product which is then converted in the same way to uric acid with the formation of hydrogen peroxide (Nelson et al., 2001).



1.7             Lipid Peroxidation


Lipid peroxidation is theintroduction of a functional group containing twocatenated oxygen atoms, O-O, into unsaturated fatty acidsin a free radical reaction (Wang, 2005). Polyunsaturatedfatty acids susceptible to free radical attack are initiatedby the formation of a carbon-centered radical by theabstraction of a hydrogen atom at one of the double bondsof the lipid. Lipid peroxidation is also one of major causesof quality deterioration during the storage of fats, oils orother lipid-rich foods (Wang, 2005). Lipid peroxidation isthe most extensively studied manifestation of oxygenactivation in biology. It is broadly defined as “oxidative deterioration of polyunsaturated fatty acids(PUFAs)” (Maneesh and Jayalekshmi,2006). Lipids when reacted with freeradicals can undergo the highly damaging chain reactionof lipid peroxidation leading to both direct and indirecteffects (Devasagayam etal., 2004).


Peroxidation of lipids is a binding process connectedwith the formation of aldehydes (Niedworok and Bielaszka,2007). Malondialdehyde(MDA) is the endproduct of lipid peroxidation, a good marker of freeradical-mediated damage and oxidative stress (Atip et al., 2010).


1.7.1Lipid peroxidation and the immune system


Lipid peroxide is a very important compound formed by the chain reaction. Non radical lipids are converted to radicals by species such as O2-, -OH, NO-and other reactive oxygen species (ROS). When the free radical removes hydrogen atom, it leaves behind an unpaired electron in the lipids (Niki et al., 2005). This in turn leads to a chain reaction by reacting with other biomolecules. The reaction is represented below:

L-H + -OH  H2O + L.

Lipid peroxidation results into a chain reaction and damages the various other molecules finally leading to cell damage. Upon lipid peroxidation, a variety of products are formed depending on the type of lipids. These products are malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), lipid hydroperoxide (LOOH), isoprostanes, conjugated dienes, lipid-DNA adduct, lipid-protein adduct, lipofuscin pigments and exhaled gases (Devasagayamet al., 2004).Malondialdehyde can react with phospholipids, nucleic acids and the free amino group of proteins leading to structural modification, which induces dysfunction of the immune system (Leet al., 2004).


Fig.4: Lipid peroxidation and generation of free radicals (Arora et al., 2013)


Lipid peroxidation is generally linked with reduced tolerance among the T-cells for the self molecules due to the general processes, infections and diseases like diabetes.



1.8 Mineral Elements


Minerals are inorganic substances present in all body tissues and fluids. Their presence is necessary for the maintenance of certain physicochemical processes which are essential to life. Although they yield no energy, they have important roles to play in many body activities (Malhotra, 1998; Erovbetine, 2003).


Every form of living matter requires these elements or minerals for the normal life processes (Hays and Swenson 1985; Ozcan, 2003). Minerals may be broadly classified as micro and macro elements on the basis of the amount in which they are required by the body (Murray et      al., 2000). The third category is ultra-trace elements. The macro-minerals include calcium, phosphorus, sodium and chloride, while the micro elements include iron, copper, cobalt, potassium, magnesium, iodine, zinc and manganese (Erovbetine, 2003).The micronutrients deficiencies which are of greatest public health significance are iron deficiency, causing varying degrees of impairment in cognitive performance, lowered work capacity, lowered immunity to infections, and pregnancy complications e.g. reduced psychomotor skills (Batra and Seth, 2002). The importance of mineral elements in human, animal and plant nutrition has been well recognized (Underwood, 1971; Darby, 1976). The trace elements are essential components of enzyme systems. Simple or conditional deficiencies of mineral elements therefore have profound effects on metabolism and tissues structure. The significance of the mineral elements in humans, animals and plant nutrition cannot be over-emphasized.



1.8.1    Biochemistry and Functions of Some Mineral Elements


The basic functions performed by the minerals are:


·        They are structural components of body tissues.


·        They are involved in the maintenance of acid-base balance and in the regulation of body fluids.


·        They transport gases and also help in muscle contraction (Murray et al., 2000) Knowledge of the importance of the mineral elements in plants is essential as the global trend in nutrition and medicine is shifting towards consumption of plant food (fruits and vegetables) and medicinal plants (phytomedicine). The plant kingdom is reported to be rich in large numbers ofsubstances beneficial to both human and animal health (Igile, 1995; Nwadiaro and Nwachukwu, 2007; Ogbonna et al., 2007; Soetan, 2008).


1.8.2    Calcium (Ca)


Calcium functions as a constituent of bones and teeth and in regulation of nerve and muscle functions. In blood coagulation, calcium activates the conversion of prothrombin to thrombin and also takes part in milk curdling. It plays a vital role in enzyme activation. Calcium activates large numbers of enzymes such as adenosine triphosphatase (ATPase), succinate dehydrogenase, lipase. It is also required in muscle contraction, membrane permeability, normal transmission of nerve impulses and in neuromuscular excitability. Reduced extra-cellular blood calcium increases the irritability of nerve tissue and very low levels may cause spontaneous discharges of nerve impulses leading to tetany and convulsions (Hays and Swenson, 1985; Malhotra, 1998; Murray et al., 2000).Calcium deficiency causes osteomalacia in adults and rickets in children (Malhotra, 1998,) and also affects both children and adult dentition (Murray, 2000).



1.8.3    Iron (Fe)


Iron is involved in the formation of haemoglobin which aids in the transportation of oxygen in cellular respiration. It functions as essential component of enzymes involved in biological oxidation such as cytochromes C, c1, Al, (Malhotra, 1998).Iron is involved in synthesis and packaging of neurotransmitters, their uptake and degradation into other iron-containing proteins which may directly or indirectly alter brain functioning (Beard, 2001). Iron is transported as transferrin, stored as ferritin or haemosiderin and it is lost in sloughed cells and by bleeding (Murray et al., 2000).



1.8.4    Zinc (Zn)


Zinc is distributed widely in plants and animal tissues and occurs in all living cells. It functions as a cofactor and is a constituent of many enzymes such as lactate dehydrogenase, alkaline phosphatase, carbonic anhydrase, alcohol dehydrogenase, glutamate dehydrogenase, DNA and RNA polymerase, etc. Zinc dependent enzymes are involved in macronutrient metabolism and cell replication (Hays and Swenson 1985). Vitamins A and E metabolism and bioavailability are dependent on zinc status (Szabo et al., 1999).


In humans, deficiency disease or symptoms include hypogonadism, growth failure, impaired wound healing, decreased taste and smell activity secondary to acrodermatitis enteropathica and parenteral nutrition (Murray et al., 2000).


Toxicity disease of zinc in humans includes gastrointestinal irritation, vomiting, decreased immune functions and a reduction in high density lipoprotein (HDL) cholesterol. High dietary levels of zinc are required in the presence of phytic acid to prevent parakeratosis and allow for normal growth (Sidhu et al., 2004).


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




1.9             Telfaira occidentalis (Fluted Pumpkin)

 Telfairia occidentalis (fluted pumpkin) is an important dioecious vegetable crop belonging to the family Cucurbitaceae (Akoroda, 1990)


The vegetable is widely grown in the tropics, majorly in South Eastern Nigeria where it is believed to have originated from. As a pot herb, the leaves are rich sources of protein, carbohydrate, iron, saponins, tannins and phytic acid and therefore a good food and medicinal source (Akaowo et al., 2000). The young shoots and leaves of the female plant form the main ingredients in edikan ikong, a soup favoured by the people of Cross River and Akwa Ibom States of Southern Nigeria (Aregheore, 2012).


Telfaira occidentalis has different traditional names. Among the Igbos, it is known as ugu, aporoko in Yoruba, ubong in Efik, umee in Urhobo and umeke in Edo (Akoroda, 1990;Kayode and Kayode2011).



1.9.1 Medicinal and nutritional properties of Telfairia occidentalis


The leaves have a pleasant taste and are rich sources of protein, oil, vitamins and minerals (Oboh et al., 2006). They enhance, nourishe, protect and heal the body. The leaves are low in crude fibre but rich sources of folic acid, calcium, zinc, potassium, cobalt, copper, iron and vitamins A, C and K(Ajibade et al., 2006). Leaves of fluted pumpkin are cheap nitrogen and mineral sources (Aregheore, 2012). The leaves of Telfaira occidentalis are also rich in mineralssuch as thiamine, riboflavin and nicotinamide (Kayode and Kayode 2011). Young leaves also possess high levels of magnesium and iron(Akaowo et al., 2000) and can prevent and eliminate anaemia (Ajibade et al., 2006). The leaves have an excellent proportion of essential amino acids to total nitrogen. Consumption of the leaves assists in combating diseases (Kayode and Kayode, 2011).


The leaves contain a considerable amount of anti-nutritive factors like high level of tannic acid and saponin. Akaowo et al.,(2000) reported that the young leaves often preferred for human consumption contain high cyanide (6.1 mg/100g) and tannin (4.6 mg/100g) than older ones.

Many researchers have observed free radical scavenging ability and antioxidant property ofTelfairia occidentalis. The darkish green leaves of T. occidentalis and extractsfrom the leaves have been found to suppress or prevent the production of free radicals and scavenge already produced free radicals, lower lipid peroxidation status and elevate antioxidant enzymes such as superoxide dismutase and catalasein vitro and in vivo (Oboh et al.,2006; Nwanna and Oboh 2007; Adaramonye et al., 2009; Kayode et al., 2010).


1.10         Tectona grandis Linn (Teak)


Tectona grandis Linn (TG) commonly known as 'Teak' is a large deciduous tree, 20-35m in height with light brown bark which belongs to the family, Verbenaceae. The leaves are 30-60 by 15-30cm, simple, opposite, broadly elliptical or obovate, acute or acuminate. It possess minute glandular dots, main nerves 8-10 pairs with 2 or 3large branches near the edge of the leaf joined by numerous parallel transverse veins (Switi and Mohan, 2011).


Plant Taxonomy


Kingdom : Plantae – Plants


Subkingdom :Tracheobionta – Vascular plants


Super division : Spermatophyta – Seed plants


Division :Magnoliophyta – Flowering plants


Class :Magnoliopsida – Dicotyledons


Subclass : Asteridae


Order : Lamiales


Family :Verbenaceae – Verbena family


Genus :Tectona L. f. – tectona


Species :grandis L. f. – teak


Botanical Name: Tectona grandis Linn





1.10.1 Medicinal importance of Tectona grandis


Tectona grandis Linn is a medicinally important plant. Various parts ofthis plant are used to treat many kinds of diseases (Khare, 2007). Oil obtained from seeds promotes the growth of hair and is useful for treatingeczema, ringworm and to check scabies (Krishna et al., 2014).Bark extract is claimed to be effective in treating conditions such as bronchitis, constipation,hyperacidity, dysentery, verminosis, burning sensation, diabetes, leprosy, skin diseases, leucoderma, headache, piles, indigestion and as worm expeller (Kirtikar and Basu, 2006; Krishna et al., 2014). Extracts from the flowers are used to treat urinary tract discharge, indigestion, dipsia, skin diseases, diabetes and as diuretic and anti-inflammatory agent. Infusion of flowers is taken in congestion of liver (Switi and Mohan, 1988).


Fig. 6: The picture of leaves of Tectona grandis Linn


All the parts of the plant seeds, flowers, fruits, wood, bark,roots, and leaves are useful either alone or along with other plants formany applications(Krishna,2006; Majumdaret al.,


2007).Chemical constituents reported in plant includeQuinones,Steroidal compounds, Glycosides,Phenolic acids and Flavonoids. Tectona grandis leaf is also reported to contain carbohydrates,alkaloids, tannins, sterols, saponins, proteins, calcium, phosphorus,crude fiber and also contain yellowish-brown or reddish dye(Goswami et al., 2009)



1.11         Statement of the Problem


The rise in immunological disorders confronting mankind today is alarming (Naga and Rajeshwari, 2014). This rise is due to different etiologies including environmental and nutritional habits. Inappropriate immunity has been shown as a common etiology in an ever-growing array of pathological processes including infections, aging, cancer and a variety of disorders of various organs (Peakman and Vergani, 1997). Most disease conditions are known to stem from or are a result of increased free radical generation and abnormal immune responses by the host (Halliwell, 1992).


HIV/AIDS (essentially an immune disorder) continues to ravage the global population especially in third world countries. No cure has been found yet and the only available therapy is to manage the condition with view to mitigating the devastating effects. Finding plants and drugs that will ameliorate the deleterious effect of HIV on T-Helper cells has remained the focus of several scientific researches. Till date, there is only a modest body of knowledge about the nutraceutical active compounds in common foods and their roles in promoting human health by immune-modulation (Tsai et al., 2011). The search for safe and potent immune modulating substances preferably with synergistic antioxidant activities continues to attract great research interest (Wang et al., 2004; Singh, 2009). This work therefore aimed at investigating the possible immunomodulating effect of Telfairia occidentalis and Tectona grandis and their possible contribution to solving the above stated problem.



1.12         Justification of the Study


Immune-modulation is a process which alters the immune system of an organism by interfering with its functions. The function and the efficacy of the immune system may be influenced by many exogenous factors like food and pharmaceuticals, stress (physical and psychological) and hormones (Dhasarathanet al., 2010; Vinothapooshan and Sundar, 2011). 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). Immunostimulatory therapy is long recognized as an alternative to conventional chemotherapy for a variety of disease conditions involving the impaired immune response of the host (Ganju et al., 2003). In healthy individuals, immune-stimulants are desirable and expected to serve as prophylactic or promotive agent by enhancing basal levels of immune response (Agrawal and Singh, 1999).Immune-modulators are becoming very popular in the worldwide natural health industry as people are becoming increasingly aware of the importance of a healthy immune system and that modulation of immune responses through stimulation or suppression may help in maintaining a disease-free state(Agrawal et al., 2010). In addition, it has been established that most pharmacologic activities are related to the immunomodulatory and antioxidant activities of plant secondary metabolites (Okonji et al., 1999). Thereis no doubt that immune-modulators hold great promise for control and prevention of infections but is yet to be fully exploited (Nworu, 2007)



1.13         Rationale for the Study


Fluted pumpkin leaves as rich sources of protein, oil, vitamins and minerals, enhance, nourish, protect and heal the body (Oboh et al., 2006). The leaves are low in crude fibre but are rich sources of folic acid, calcium, zinc, potassium, cobalt, copper, iron, vitamins A, C and K (Ajibade et al., 2006) and are cheap nitrogen and mineral sources (Aregheore, 2012).


Many researchers have reported free radical scavenging ability and antioxidant property ofTelfairia occidentalis (Oboh et al., 2006; Nwanna and Oboh, 2007; Adaramonye et al., 2009; Kayode et al., 2010).


Tectona grandis on the other hand is a medicinally important plant. Various parts of this plant are used for treating many disease conditions such as bronchitis, constipation, hyperacidity, dysentery, burning sensation, diabetes, leprosy, skin diseases, leucoderma, headache, piles, indigestion, and as worm expeller (Kirtikar and Basu, 2000; Krishnan, 2006; Khare, 2007) and have been shown to possess anti-inflammatory activities (Switi and Mohan 2011). Besides, the fresh leaves are used to treat anaemic conditions in traditional medicine in some parts of Enugu State of South Eastern Nigeria. Both plants are known to be rich in phytochemicals and antioxidant compounds which are vital sources of immune-modulating agents(Goswamiet al., 2009).Telfairia occidentalis Hook F and Tectona grandis Linn are well known for haematinic and other medicinal properties (Nwanna and Oboh, 2007; Kayode et al., 2010). However there is no documented evidence of any serious investigation of their immune-modulatory effects (Jaybhayeet al., 2010), hence this study. It is hoped that immune-modulating agents contained in these plants will be of immense therapeutic benefit to persons infected with some pathologic challenge to their immune system due to infections and diseases.


1.14         Aim of the Study


To investigate the immune-modulatory and possible synergistic antioxidant effects of the crude extracts and different fractions of Telfairia occidentalis Hook F and Tectona grandis Linn on normal and immune-compromised rats.



1.15         Specific Objectives of the Study


1.  To evaluate the effect of the extracts on some key hematological parameters.


2.    To investigate the effect of the extracts on humoral antibody titre and delayed type hypersensitivity (DTH) reactions.


3.  To investigatein vivo antioxidant and free radical scavenging effect of the extracts.


4.  To investigate the effect of the extracts on serum micro-nutrient levels in rats.


5.   To investigate stimulation of lymphocyte proliferation and cytokine production by both T-Helper 1 and T-Helper 2cells by the extracts.


6.  To quantitate the stimulation of CD4+ lymphocyte sub-population by the extracts.


7.    To compare the effect of the extracts to that of a standard immune-modulatory drug, Dynamogen.


8.  To characterize the phytochemicals responsible for the bioactivity of the extracts using GC-MS, IR and NMR techniques.


9.     To compare the effects produced by extracts of Telfairia occidentalis Hook F with thatofTectona grandis Linn.




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