ABSTRACT
In the present study, the phytochemical composition, immunomodulatory, leukocyte mobilization, haematological and antihepatotoxic effects of the aqueous extract of Senna mimosoides leaves were evaluated. The study also covered the effect of the extract on the activity of lactase and the assessment of the damaging effect of carbon tetrachloride (CCl4) and ameliorative effect of the extract on liver tissue using histopathological technique. This study was aimed at validating the traditional use of S. mimosoides leaves in folklore medicine to treat breast milk toxicity in neonates by elucidating its immunological and biochemical nature. The qualitative and quantitative phytochemical composition showed the presence of
2.67 ± 0.0013 mg of flavonoids; 3.43 ± 0.0028 mg of alkaloids; 1.97 ± 0.0030 mg of saponin; 2.32 ± 0.0032 mg of terpenoids; 0.86 ± 0.0023 mg of steroid; 3.61 ± 0.0025 mg of phenol; 8.31 ± 0.0032 mg of reducing sugar; 4.75 ± 0.0034 mg of tannin; 1.61 ± 0.0031 mg of cyanide; 2.75 ± 0.0029 mg of glycoside and 4.68 ± 0.0033 mg of soluble carbohydrates for every 100 g of the extract. For the animal model experiment, one hundred and thirty (130) albino rats were used. The experimental design was divided into four (4) phases containing five (5) groups of five (5) rats in each group. Rats in group A (control) were administered 0.2 ml of normal saline; rats in groups B, C and D were treated with 50, 100 and 250 mg/kg of the aqueous extract of S. mimosoides leaves respectively; group E rats received levamisol or silymarin (standard drugs) while group F rats were treated with carbon tetrachloride (CCl4) only. Administration of 50, 100 and 250 mg/kg of the extract resulted in a dose-dependent significant (p < 0.05) increase in primary antibody titre with a value of 6, 8, 13, and secondary antibody titre with a value of 11, 26, 34. Delayed type hypersensitivity (DTH) response shows that the extract produced a dose- and time-dependent increase in footpad swelling of the rats. The extract (50, 100 and 250 mg/kg) and levamisol (25 mg/kg) at 24 hr after challenge, significantly (p < 0.05) boosted DTH reactions observed respectively as
1.412, 1.504, 1.816 and 1.827 mm difference in thickness of footpad before challenge and 24 hr after challenge while the control ellicited a non-significant (p > 0.05) increase with a difference of 0.614 mm. At 48 hr after challenge, there was an additional increase in footpad swelling observed as 1.908, 1.918, 2.304 and 2.326 mm for the extract and levamisol respectively. The humoural antibody (HA) titre and DTH response compare well with that of levamisol, a standard immunostimulatory drug, at 25 mg/kg. The total leukocyte count of the groups treated with different concentrations of extract increased in a dose-dependent manner while the group treated with indomethacin decreased significantly (p < 0.05) compared with control. The percentage packed cell volume (PCV) for group B, before and after treatment with cyclophosphamide (CP) and later with (50 mg/kg) was 38.8 ± 1.30, 19.4 ± 0.55 and 34.4 ± 0.55 respectively. Groups C, D, and E showed the same trend but in the control group decrease by CP was not reversed. In the control, percentage PCV before and after CP and then extract was 35.8 ± 0.45, 19.4 ± 0.55 and 19.8 ± 1.09 respectively. The same trend was observed in haemoglobin concentration, white blood cell count, red blood cell count and its indices. There was increase in serum alanine aminotransferase (ALT) activity of rats in group F (81.20 ± 0.84 IU/L) after CCl4 administration as compared to the normal control A (53.00 ± 1.00 IU/L). The extract (50, 100, 250 mg/kg) and silymarin (25 mg/kg) caused a significant (p < 0.05) decrease in the activity of ALT (65.00 ± 1.58, 59.20 ± 0.84, 55.20 ± 1.30 and 57.00 ± 1.00 IU/L) respectively. The levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, malondialdehyde, iron, phosphate followed the same trend as ALT compared to control. Administration of CCl4 decreased the level of reduced glutathione in group F (2.21 ± 0.239 mMol/g tissue). However, treatment with different concentrations of the extract and levamisol augmented this decrease (3.08 ± 0.093, 4.17 ± 0.241, 5.16 ± 0.193 and 4.97 ± 0.273 mMol/g tissue) respectively. Activities of glutathione s-transferase, glutathione peroxidase, catalase, superoxide dismutase and concentrations of sodium, magnesium, potassium, calcium, zinc and selenim showed the same trend. Histopathological studies showed that the extract and levamisol ameliorated centrilobular degeneration of the liver tissues induced by CCl4. Moreover, the extract exhibited higher significant (p < 0.05) activity of lactase in a dose-dependent manner when compared to the control. At 10, 20, 30, 40 and 50 µl, the enzyme activity were 17.187, 18.822, 20.044, 22.022 and 23.898 IU.The findings of this study show that the vase medicinally important bioactive compounds, present in this extract could be responsible for the immunostimulatory, antihepatotoxic effect, increase in lactase activity and haematological parameters. This justifies the use of this plant in folklore medicine for the treatment of diseases.
CHAPTER ONE
INTRODUCTION
Plants are known to contain a variety of secondary metabolites. These secondary metabolites or bioactive compounds have definite physiological effects on the human system. According to Yadav and Agarwala (2011), approximately 25 percent of all prescribed medicines today are substances derived from plants. Interestingly, many phytochemicals have been discovered and even isolated from a variety of medicinal plants. However, many more of them are yet to be exploited for clinical use. Phytochemical analysis of plants is importante due to the need for alternative drugs of plant origin, made imperative by the high cost of synthetic drugs. These secondary plant metabolites extractable by various solvents exhibit varied biochemical and pharmacological actions in animals when ingested (Nwogu et al., 2008).
The use of Senna mimosoides in folklore medicine, precisely in Ukehe, Nsukka, to treat oedema and breastmilk toxicity in neonates was the rationale behind this work. The anti- inflammatory capacity of the leaf extract of Senna mimosoides and its mechanism of action has been reported by Ekwueme et al. (2011a,b).In Nsukka, immediatly after delivery, breastmilk is usually dropped on the leaves of cocoyam or on ants to check its toxicity.Toxic breastmilk usually burns the leaves of the cocoyam or kills any ants it comes in contact with. The prevalence of industries predisposes mothers to chemicals that might accumulate in breast milk. In this study, the immunomodulatory activity and anti-hepatotoxic effect of the leaf extract of S. mimosoideswas investigated because they are the basic mechanism used by the body to prevent or cure diseases. Moreover, the effect of the leaf extract on the activity of lactase,the enzyme that catalyzes the hydrolysis of lactose which is the only carbohydrate present in breast milk was assayed for.
1.1 Overview of the Human Immune System
Immunology is the study of the methods by which the body defends itself against infectious agents and other foreign substances in its environment (Wotherspoon, 2012). There are thousands of components to the immune system and it would appear that the immune system is far more complicated than necessary for achieving what is, on the surface, a simple task of eliminating a pathogenic organism or abnormal ‘self’ cells (Parkin and Cohen, 2001). However there are a number of reasons for this complexity, including the desirability of eliminating pathogens without causing damage to the host. Getting rid of a pathogen or dead
host cells is theoretically easy, but eliminating these without damaging the host is much more complicated. As a consequence of this dynamic complexity, the immune system is able to generate a tremendous variety of cells and molecules capable of specifically recognising and eliminating an apparently limitless variety of foreign invaders, in addition to the recognition and destruction of abnormal cells (Parkin and Cohen, 2001). Once a foreign protein, microorganism (e.g., bacterium, fungus or virus) or abnormal cell is recognised, the immune system enlists the participation of a variety of cells and molecules to mount an appropriate effector response to eliminate or neutralise them (Parkin and Cohen, 2001). Later exposure to the same foreign organism induces a memory response, characterised by a heightened immune reactivity, which serves to eliminate the microbial pathogen, prevent disease and protect against the development of some tumour cells.
1.2The Cells of the Immune System
1.2.1 T Lymphocytes
T-lymphocytes do not produce antibody molecules rather they directly attack foreign antigens such as viruses, fungi, or transplanted tissues (Kruisbeek et al., 2004). One T-cell class carries the CD8 molecule which binds to MHC class I while the other carries the CD4 molecule which binds to MHC class II. T-lymphocytes based on their function are grouped into killer or cytotoxic T-lymphocytes, helper T-lymphocytes, and regulatory T-lymphocytes. T cells displaying CD4+ generally function as TH cells, whereas those displaying CD8+ function as TC cells.Killer, or cytotoxic, T-lymphocytesperform the actual destruction of the invading microorganism (Luckashenaket al., 2008). They do this by migrating to the site of an infection or the transplanted tissues, directly binding to their target and killing it by lysing.
The helper T-lymphocyte and “helps” or enhances the function of B-lymphocytes, causing them to produce quickly more antibodies and to switch from the production of IgM to IgG and IgA and and also assist killer T-lymphocytes in their attack on foreign substances (Parkin and Cohen, 2001). Activation of TH cell makes it an effector cell that secretes various cytokines (O’Keefe et al., 2002) that plays an important role in activating B cells, TC cells, macrophages, and various other T cells, and initiate the delayed type hypersensitivity (DTH) response (Parkin and Cohen, 2001).Regulatory T-lymphocytes suppress or turn off other T- lymphocytes. Without regulatory cells, the immune system would keep working even after an infection had been cured and overreact to the infection (Vignali et al., 2008).
1.2.2 B-lymphocytes
B-lymphocytes (sometimes called B-cells) are specialized cells of the immune system whose major function is to produce antibodies (also called immunoglobulins or gammaglobulins) (Leen et al., 2013). Antibodies are complex molecules (glycoproteins) that have the property of combining specifically to the antigen that induced its formation. Antibodies are catholic in their recognition; they can recognize free proteins, in solution; proteins displayed on cell walls or membranes; and proteins within higher-order structures, such as viral capsids. When B-lymphocytes are stimulated by antigens, they respond by maturing into plasma cells which are the cells that actually produce the antibodies. These antibodies then find their way into the bloodstream, tissues, respiratory secretions, intestinal secretions, and even tears. The resulting antibodies bind to the invading pathogen, marking it for destruction by killer T- lymphocytes by a process called antibody dependent cell cytotoxicity (ADCC) (Clemenceau,
2008). Antibodies also mark cells for phagocytosis by neutrophils and other phagocytic cells by a process called opsonisation. Most of the daughter cells produced by B cell activation die within a few weeks but a proportion of them recirculate in the body for many years as memory cells. If they are reintroduced to the same antigen that elicited an initial response, they rapidly become reactivated and produce antigen-specific antibody (Leen et al., 2013). There are five distinct classes of antibody, based on the type of heavy chain involved; Immunoglobulin G (IgG); Immunoglobulin A (IgA); Immunoglobulin M (IgM); Immunoglobulin E (IgE); Immunoglobulin D (IgD).
The IgG class is the only class of immunoglobulins which crosses the placenta and passes immunity from the mother to the newborn (Walter and Theil, 2011). Antibodies of the IgA fraction are produced near mucus membranes and find their way into secretions such as tears, bile, saliva, and mucus since it can be transported across, where they protect against infection in the respiratory tract and intestines. Antibodies of the IgM class are the first antibodies formed in response to infection. They are important in protection during the early days of an infection. Antibodies of the IgE class are responsible for allergic reactions. IgE sensitizes specialized ‘mast’ cells, important in protecting against parasitic infections.
1.2.3 Natural killer (NK) Cells
NK cells are large, granular lymphocytes that are capable of lysing or killing infected or tumour cells without overt antigenic stimulation or recognition (recruiting specific immune response) (Parkin and Cohen, 2001). These cells can be considered complementary to
cytotoxic T lymphocytes (CTLs). Many viruses attempt to circumvent CTL recognition by preventing the MHC molecule from reaching the cell surface – and here natural killer (NK) cells step into the breach. These cells do not recognize specific foreign antigen, instead being activated by the absence of MHC molecules on a cell’s surface, activated NK cells destroy susceptible target cells by inoculating a protein named perforin into the target cell membrane; perforin molecules assemble in the membrane to form a pore, through which other toxic molecules can flow into the target. NK cells are also prolific producers of the antiviral cytokine interferon g (Kim et al., 2011). At the sites of inflammation, activated macrophages produces IL-12 which stimulate NK cells to produce IFN.
1.2.4 Monocyte and Macrophages
Monocytes which make up 2-8% of the WBCs leave circulation and enter tissue, as macrophages. There are two types of macrophages, one that wander in the tissue spaces and the other that are fixed to vascular endothelium of liver, spleen, lymph node and other tissue (Parkin Cohen, 2001). Macrophages are large leukocytes derived from monocytes that function in phagocytosis, antigen processing and presentation, secretion of cytokines and antibody-dependent cell-mediated cytotoxicity (ADCC). Functions of macrophage include killing of microbes, infected cells, and tumor cells, secretion of immunomodulatory cytokines, antigen processing and presentation to T cells. Macrophages respond to infections as quickly as neutrophils but persist much longer; hence they are dominant effector cells in the later stage of infection.
1.2.5 Antigen-Presenting Cells (APCs)
Specifically, APCs are any cells that can process and present antigenic peptides in association with class II MHC molecules on the surface of antigen-presenting cells or altered self-cells (Accolla and Tosi, 2012).These specialised cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by two properties: they express class II MHC molecules on their membrane, and they are able to deliver a co-stimulatory signal that is necessary for TH-cell activation (Kuby, 1997). In the presence of soluble antigen, TH cells primed by dendritic cells can interact with B cells and stimulate antigen-specific antibody production (Girolamo et al., 2008). Dendritic cells are equally important in priming CD8+ or TC cells. Interestingly, dendritic cells can directly induce cytotoxic TC cell proliferation with help from TH cells. Antigen-presenting cells (APC) can also elicit a local rapid reaction or cascade of events that triggers the specific-immune responses.
1.2.6 Phagocytes
Phagocytes are specialized cells of the immune system whose primary function is to ingest and kill microorganisms. There are several different types of phagocytic cells. Polymorphonuclear leukocytes (neutrophils or granulocytes) are found in the bloodstream and can migrate into sites of infection within a matter of minutes. It is this phagocytic cell that increases in number in the bloodstream during infection and is in large part responsible for an elevated white blood cell count during infection. Polymorphs play a major role in controling many infections, travelling rapidly to the affected site, assisting in the recruitment of other immune responses, and engulfing the microbes and other debris (Wang, et al., 2006). It is also the phagocytic cell that leaves the bloodstream and accumulates in the tissues during the first few hours of infection, and is responsible for the formation of “pus” (Dale et al.,
2008). Monocytes, another type of phagocytic cell, are also found circulating in the bloodstream.
1.2.7 Neutrophils
Neutrophils are the most abundant leukocytes in our circulation and become rapidly mobilized to eliminate microbes and necrotic cells in areas of infection or inflammation (Nathan, 2006). Despite having a brief half-life and lacking proliferative potential, neutrophils have the ability to synthesize and release immunoregulatory factors, thereby helping the recruitment of DCs and monocytes that not only complete innate clearance of invading microbes, but also initiate more specific adaptive immune responses (Mantovani et al., 2011). Neutrophils are characterized by the presence of cytoplasmic granules primary (or azurophilic) granules which predominates in early stages of neutrophil maturation and are less capable of exocytosis than secondary (or specific) granules, which are generated in later developmental stages. Primary granules contain myeloperoxidase (MPO), which is important for the digestion of phagocytosed material (Mantovani et al., 2011) while secondary granules contain lactoferrin and gelatinase, which degrade the extracellular matrix, exert antimicrobial activity and initiate inflammation.
In addition to undergoing degranulation, neutrophils generate a respiratory burst by activating an enzymatic complex known as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which generates reactive oxygen species involved in microbial killing (Puga et al.,
2012). Moreover, neutrophils can also form neutrophil extracellular traps (NETs), which are cellular projections capable of trapping and killing bacteria. These structures contain
decondensed chromatin embedded with cytoplasmic and granular proteins with powerful antimicrobial functions, including serin proteases and antimicrobial peptides such as cathelicidin (Brinkmann et al., 2004).
1.2.8 Basophils and Mast Cells
Mast cells are tissue-resident leukocytes very similar to basophils. There are at least two populations of mast cells, based on the enzymes they contain and their tissue location (Parkin and Cohen, 2001). T mast cells (mucosal mast cells) contain only trypsin, whereas connective tissue mast cells contain both trypsin and chymotrypsin. Mast cells and basophils bear high- affinity receptors for IgE FcRI (CD23) which rapidly absorbs any local IgE (Puga et al.,
2012). Crosslinking of these receptors by the binding of antigen to IgE leads to degranulation and release of preformed mediators, such as the vasoactive amines, histamine and serotonin. Membrane derived mediators such as leucotrienes B4, C4, D4 and E4, prostaglandins and platelet activating factor are also produced leading to increased vascular permeability, bronchoconstriction, and induction of an inflammatory response.
Basophils produce histamine and other vasoactive compounds, immunomodulating factors such as platelet-activating factor (PAF), leukotriene C4, granzyme B and retinoic acid as well as antibody-inducing and Th2-differentiating cytokines, including IL-4, IL-6 and IL-
13(Karasuyama et al., 2011). Among basophil-tropic cytokines, IL-3 enhances basophil recruitment into lymphoid tissues, augments basophil secretion of IL-4 and promotes basophil expansion after parasite infection. However, some studies show that IL-3 is not required for the maintenance of basophils in vivo, probably because this function is also covered by the IL-7-like cytokine thymic stromal lymphopoietin (TSLP). Basophils release IL-4 and facilitate the differentiation of Th2 cells producing IL-4 in response to signals from IgE-binding antigens, cytokines (IL-3, GM-CSF, IL-33 or IL-18), microbial receptors (TLR2 and TLR4), and allergenic proteases (Sokol et al., 2009).
1.2.9 Eosinophils
Eosinophils, the second most frequent granulocyte subset in the circulation protects host from parasitic (particularly nematode) infections. Such infections induce antigen-specific IgE production, the antibodies coating the organism then eosinophils binds its low affinity receptors (FcRII). Eosinophils are not phagocytic, but have large granules containing major basic protein, eosinophilic cationic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin, which are highly cytotoxic when released onto the surface of organisms (Puga et
al., 2012). In recent years eosinophils have also been shown to modulate adaptive immunity as a result of their ability to up-regulate the expression of MHC-II molecules and secrete cytokines, chemokines, lipid mediators and growth factors (Puga et al., 2012).
Eosinophils modulate innate immune responses by regulating the activation of mast cells, basophils and neutrophils through MBP. In addition, eosinophils induce the expression of antigen-loading MHC-II and T cell costimulatory molecules after undergoing transendothelial migration and in the presence of appropriate cytokines (Akuthota et al.,
2010). Eosinophil production of chemokines and cytokines such as TNF, IL-4 and IL-12 not only influences the recruitment and maturation of DCs, but also induces the differentiation of Th1 and Th2 cells.
1.3Innate (Non-Specific) Immunity
Innate or non-specific immunity which refers to the basic resistance to disease that an individual is born with, provide the first line of host defence against invading microbial pathogens and also protects against some tumour cells until an acquired immune response develops (Dhasarathan et al., 2010). Innate immunity can be envisioned as comprising four types of defensive barriers: anatomic; physiologic; endocytic and phagocytic; and inflammatory (Parkin and Cohen, 2001). The physiologic barriers that contribute to innate immunity include elevated temperature (e.g., fever), pH (e.g., acidity produced in stomach and within macrophages), oxygen tension, and various soluble factors (Kuby, 1997). Thera are also soluble proteins such as lysozyme, interferons (INF) and other cytokines and complement. A central feature of the innate reaction is recruitment and activation of neutrophils at the site of infection to eradicate pathogens. During the very early stages of infection or tissue damage, there is release of cytokines from activated macrophages. Two of these, granulocyte and granulocyte-macrophage colony stimulating factors, stimulate division of myeloid precursors in the bone marrow, releasing millions of cells into the circulation and causing a characteristic neutrophil leucocytosis (Wotherspoon, 2012). To home to a site of infection, neutrophils use a multistep process involving proinflammatory mediators, adhesion molecules, chemoattractants, and chemokines (Nathan, 2006). The recruited neutrophils phagocytose organsisms by making pseudopodia (projections of cytoplasmic membrane) which form a membrane-bound vesicle (phagosome) around the particle (Parkin and Cohen,
2001). In this protected compartment killing of the organism occurs by a combination of two mechanisms. The oxygen-dependent response or respiratory burst which involves the
sequential reduction of oxygen by an NADPH oxidase leading to production of toxic oxygen metabolites, such as hydrogen peroxide, hydroxyl radicals, and singlet oxygen (Paoliello- Paschoalato et al., 2011).
1.4Adaptive Immunity
Adaptive (acquired, specific) immunity is capable of recognizing and selectively eliminating foreign microrganism and molecules. These host defences are mediated by twointerrelated and interdependent mechanisms:
Humoural immunity which primarily involves bone marrow-derived (B) lymphocytes or B-cells.
Cell-mediated (cellular) immunity which primarily involves thymus-derived (T)
lymphocytes or T-cells.
The characteristic of adaptive immunity is the use of antigen-specific receptors on T and B cells to drive targeted effector responses in two stages. First, the antigen is presented to and recognised by the antigen specific T or B cell leading to cell priming, activation, and differentiation (Parkin and Cohen, 2001). Secondly, the effector response takes place, either due to the activated T cells leaving the lymphoid tissue and homing to the disease site, or due to the release of antibody from activated B cells (plasma cells) into blood and tissue fluids, and hence to the infective focus.
Upon exposure to an antigen, specific molecules capable of recognizing only that antigen are activated, to eradicate the foreign material (Shi, 2004). Unlike nonspecific responses, the specific response has ‘memory’–when the antigen is encountered for a second time, the antigen-specific host response is much faster, and much more extensive. For this reason, in contrast to nonspecific innate immunity, antigen-specific responses are said to be ‘adaptive’.
1.5Humoural Immunity
Humoural immunity is defined in terms of the B-lymphocytes (B-cells), the antibody producing cells of the immune system. Antibodies function in concert with complement proteins that are produced in the liver and by macrophages to provide protection against bacterial and viral infections and agents that causes tumour (Gupta et al., 2008). Humoural immunity can be further classified with regard to the dependence of antibody production on T lymphocyte help: T-cell dependent and T-cell independent immunities. Each B lymphocyte is genetically programmed to produce a single specific antibody with a particular molecular shape. The shape of an antibody allows it to bind with a specific antigen when a B-cell
encounters that antigen in the bloodstream. For this purpose, each B-cell carries a “prototype” of its antibody embedded in its surface. When the matching antigen is encountered, the B-cell proliferates and differentiates, producing plasma cells which actively secrete a soluble form of the antibody (Sumen et al., 2004).
Antibodies can work in several different ways, depending largely on the form of antigen to which they react. Some functions include: Interlocking directly with toxic chemicals or toxins produced by an organism to neutralize them; coating (opsonizing) cells to make them more palatable to scavenger cells or signal their presence to “killer” lymphocytes (this last is a process known as antibody-dependent cell-mediated cytotoxicity or ADCC.); binding with antigen to secrete a lethal group of enzymes known as complement; blocking viruses from entering cells; preventing a cell (usually a virus cell) from reproducing; this function appears to act against tumor cells undergoing metastasis (Gupta et al ., 2008).
1.6Cell-Mediated Immunity (CMI)
CMI is associated with the T-lymphocytes or T-cells (thymus-derived). Various classes of T- cells have been described, such as suppressors, helpers, inducers, and cytotoxic cells (Shevach, 2000). These are divided into two categories: regulatory T-cells, which help orchestrate cell responses; and cytotoxic T-cells which directly attack body cells which are infected (by a virus) or malignant (cancerous). The most important type of regulatory T-cells are known as helper/inducer cells, sometimes abbreviated TH -cells. These are responsible for activating B cells as well as nearby natural killer cells and macrophages (Yoon and Jun,
2005). As the name implies, suppressor cells abbreviated TS act to turn off or suppress the actions of T-cells. Cytotoxic T-cells are a type of “killer cell” which, in addition to attacking malignant cells, is also responsible for rejecting tissue or organ grafts (Shevach, 2000).
Some T-cells secrete various peptide factors, referred to as lymphokines or cytokines that modulate the activity of B- and T-cells. Like antibodies, lymphokines play several different roles; many are toxins that directly attack infected cells. One of these cytokines, called tumor necrosis factor, can play an important role in cancer remission (Grivennikov and Karin,
2011). Other lymphokines, including an important one called interferon, incite macrophages to engulf tumor and virus cells and to produce cytokines of their own. Still others promote the production or maturation of additional T-cells or direct B-cells to produce antibody. T-cells are now commonly defined in terms of various membrane “antigens”, such as T-4 (or CD4) for helper/cytotoxic cells and T -8 (or CD 8) for suppressor/cytotoxic cells (Shevach, 2000).
T lymphocytes, however, need the antigen to be processed and presented to them by an APC. The T-cell antigen receptors (TCRs) recognize fragments of antigens bound to molecules of the major histocompatibility complex (MHC) on the surface of an APC. Intracellular antigens, cut into peptides in the cytosol of the APC, bind to MHC class I molecules and are recognized by CTLs, which, once activated, can directly kill a target cell. Extracellular antigens that have entered the endocytic pathway of the APC are processed there and generally presented by MHC class II molecules to T-helper cells, which, when turned on, have profound immune-regulatory effects.
1.7Mediators of the Immune System
1.7.1 Cytokines: The chemical messengers
The term cytokine covers a variety of small proteins less than 20 kDa that serve a hormone- like function in enabling cells to communicate with each other. Cytokines are small molecular weight messengers secreted by one cell to alter the behaviour of it or another cell. Cytokines send intracellular signals by binding to specific cell-surface receptors. Different cytokines can either act synergistically or antagonistically (Minich and Bland, 2008). Cytokines are produced by virtually all cells and have a wide variety of functions. The biological effect depends on the cytokine and the cell involved, but typically these molecules will affect cell activation, division, apoptosis, or movement.
1.7.2 Complement System
The complement system provides innate defense against microbial infection and is a “complement” to antibody mediated immunity. Complement system is composed of more than 35 different proteins produced by hepatocytes, macrophages and intestinal epithelial cells. Fibroblasts and intestinal epithelial cells make C1, while the liver makes C3, C6, and C9 (Glovsky et al., 2004). These proteins (circulating in the serum or membrane bound) forms a sophisticated molecular network capable of recognizing, tagging, and eliminating invading pathogen and altered host cells (e.g., apoptotic and necrotic cells) via Ab- independent mechanisms (Gupta et al., 2008). Thus, the complement system provides the first line of defense before the adaptive immune response builds up. Moreover, the complement system bridges the innate and adaptive immunity, because the activated complement components facilitate the phagocytosis of pathogens by the host’s leukocytes and initiate inflammatory reactions by recruiting and stimulating the cellular elements of the immune system (Parkin and Cohen, 2001).
In some instances, microorganisms must first combine with antibody in order to activate complement while in other cases; the microorganisms can activate complement without the need for antibody. Some components of complement send out chemical signals to attract phagocytic cells while others coats microorganisms making them more easily ingested by phagocytic cells. When the complement system is assembled on the surface of some microorganisms, a complex is created which can puncture the microorganism and cause it to burst (Cole and Morgan, 2003).
During activation, some complement components are split into two parts. The larger part of the molecule is called “b” and the smaller fragment called “a” and may diffuse away (Glovsky et al., 2004). In most cases “b” fragment binds to the surface of the cell to be lysed except C2. There are three pathways of activation namely classical pathway, lectin pathway and alternative pathway (Glovsky et al., 2004).
Although triggered by different events, and initially employing different components of the complement system, all three activation pathways converge to a single point, the production of a protein named C3 convertase (Sahu and Lambris, 2001). This leads to the activation of all three effector arms of the complement cascade. The first effector mechanism (and probably the most important) is coating (or ‘opsonization’) of pathogens with the C3b complement component; this interacts with receptors on the surface of phagocytes, encouraging pathogen engulfment. The second effector mechanism is production of a
‘membrane attack complex’, in which a monomeric protein undergoes assembly followed by insertion into the lipid membrane of the pathogen, or of the infected cell, generating a membrane-spanning pore, similar to that formed by perforin, which disrupts homeostasis (Moreno, 2000). The third aspect of complement’s effect is release of peptide inflammatory mediators which can aid in the recruitment of phagocytes and monocytes to the site of infection.
Normal host cells bear the complement receptor type 1 and decay accelerating factor, which inhibit C3 convertase and prevent progression of complement activation. However, microbes lack these molecules and are susceptible to complement (Parkin and Cohen, 2001). In addition to lysis of organisms, complement has other anti-infective functions. There is the opsonic action of C3b, the release of soluble C3a and C5a, which are anaphylatoxins and increase vascular permeability allowing proteins, such as antibody, to penetrate the tissue, and the chemotactic activity of C5a that induces an inflammatory infiltrate (Gupta et al.,
2008). Complement also has a role within the specific immune response; its activation and deposition within immune complexes helps to target these to complement-receptor bearing antigen-presenting cells, such as B lymphocytes and follicular dendritic cells.
1.8 Blood
Blood is a tissue which consists of fluid plasma in which are suspended a number of formed elements (erythrocyte, leucocyte and thrombocytes). Its primary function is to provide a link between the various organs and cells of the body, and to maintain a constant cellular environment by circulating through every tissue delivering nutrient to them and removing waste products (Yona and Jung, 2009). The blood cells exist at fairly constant levels, suggesting the existence of feedback mechanism for the cells (Guyton and Hall, 2006). Haematology offers a wide spectrum of interest and interaction in medicine and offers the unique opportunity to combine laboratory and clinical data in a rapidly changing science (Nwodo et al., 2010). The assessment of haematological parameters could be used to reveal the deleterious effect of foreign compounds including plant extracts on the blood constituents of animals. They are also used to determine possible alterations in the levels of biomolecules such as enzymes, metabolic products, haematology, normal functioning and histomorphology of the organs (Akhtar et al., 2012).
Haematological parameters, which include complete blood count-Haemoglobin, Packed cell volume, Leukocyte (total and differential), Platelet, Red blood cell, Reticulocyte and absolute indices, are all important in the diagnosis and classification of anaemia. Anaemia is the reduction in heamoglobin and hematocrit in relation to age, sex and location of individual considered (Ramin et al., 2012). The major concern of the scientific communities with regard to medicinal plants and heamatological studies focuses on the measures that can maintain a normal haematological state of being and reverse any negative haematological status associated with various anaemic conditions.
1.9The Concept of Immunomodulation
Immunomodulation is a procedure which can alter the immune system of an organism by interfering with its functions; if it results in an enhancement of immune reactions it is named as an immunostimulative drug which primarily implies stimulation of specific and non specific system, i.e. granulocytes, macrophages, complement, certain T-lymphocytes and different effector substances. Immuno-suppression implies mainly to reduce resistance against infections, stress and may occur on account of environmental or chemotherapeutic
factor. The immune responses through stimulation or suppression may help in maintaining a disease-free state. Agents that activate host defense mechanisms in the presence of an impaired immune responsiveness can provide supportive therapy to conventional chemotherapy.
1.10 Cyclophosphamide (CP)
2-[Bis(2-chloroethyl)amino] tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide, CP is an alkylating agent that is frequently used as an antineoplastic drug (Sulkowska et al., 2002). Alkylation of CP which involves loss of a chlorine molecule and replacement with -CH3 produces intra- and interstrand DNA crosslinks inactivating DNA. The crosslinks are responsible for the cytotoxicity of the cyclophosphamide. CP is a prodrug that requires activation by the cytochrome P450 enzyme system to form its pharmacologically active metabolite, 4- hydroxycyclophosphamide and its tautomer aldophosphamide in the liver (De Jounge et al., 2005).
The unique pharmacology of high-dose CP accounts for its potent immunosuppressive properties and ability to spare haematopoietic stem cells. Lymphoid cells, including natural killer cells, B and T lymphocytes, have low levels of aldehyde dehydrogenase and are rapidly killed by high doses of CP (Brodsky, 2002). However, primitive haematopoietic stem cells possess high levels of aldehyde dehydrogenase rendering them highly resistant to cyclophosphamide (Brodsky, 2002). Therefore, high-dose cyclophosphamide is highly immunosuppressive, but not myeloablative; endogenous haematopoietic stem cells will reconstitute haematopoiesis without the need for a stem cell graft.
1.10.1 Metabolism of Cyclophosphamide
Cyclophosphamide is activated by hepatic microsomal mixed function oxidases cytochrome P450 to form 4–hydroxycyclophosphamide (4-OHCP), which exists in equilibrium with its tautomer aldophosphamide (AldoCP). 4-OHCP is very unstable, readily diffuses in to cells and spontaneously decomposes into phosphoramide mustard (PM) by ß elimination of acrolein. PM is an active alkylating species which is responsible for alkylating effect of CP. Acrolein is an unwanted by product which may enhance CP-induce cell damage, possibly by depletion of cellular glutathione by conjugation (Blomgren and Hallstrom, 1991). The mechanism of action of alkylating agents consists in the conversion of an active hydrogen atom from the biologically active molecules (DNA, RNA, enzymes, mucopolysaccharides).
The alkylation concerns carboxyl groups, amino-terminals, phosphate groups and others. The alkylation of the biologically active molecules causes an impairment of their functions.
1.10.2 Mechanism of Action of Cyclophosphamide
Following activation of CP in the liver, multiple metabolites appear in the circulation with varying degrees of immunosuppressive action and toxicity (McDonald et al., 2003). Although direct toxicity to immunocompetent cells is probably the major mechanism of immunosuppression, CP is also immunomodulatory in T cells. The immune effects of CP differ depending on the dose, route of administration, and duration of CP therapy. Frequently encountered toxicities include bone marrow suppression and mucosal lining abnormalities. Because cyclophosphamide metabolites are excreted in the urine, hemorrhagic cystitis and bladder cancer are also prominent complications (Choonget al., 2000).
MFO-mediated metabolism of CP is an important, but not exclusive pathway to bioactivate various xenobiotics (Zhou et al., 2003). The involvement of other metabolic pathways, such as cooxidation via prostaglandin H synthase (PHS) in the toxicity of CP has been postulated. In contrast to MFO-s, found in the highest concentrations in the liver, PHS and lipoxygenase activities are relatively high in the lung and bladder, sites of major CP-induced toxicity (Hayes et al., 2005). 24 hr after CP administration there will be marked polymorphism of the mitochondria and condensation of their matrix, segmentary blurring of the structure of the surrounding membranes, the presence of osmophilic intramitochondrial bodies and paracrystalline structures usually arranged along the organelles (Zhanget al., 2006). Golgi complexes will be stimulated. The rough endoplasmic reticulum will be focally degranulated, while the smooth endoplasmic reticulum appeares considerably proliferated.
1.11 Levamisole
Levamisole is a synthetic phenylimidazothiazole that is undergoing clinical evaluation as an antineoplastic agent. Although originally used as an antihelminthic drug, oncological interest in this drug stems from early reports demonstrating restorative effects of levamisole on suppressed immune responses, and antitumor activity in animal tumor models (Shah et al.,
2011). Levamisole has been shown to improve immunitary defences and delayed type hypersensitivity in immunodepressed individuals, to restore T helper and T suppressor cell activity in old mice and to evoke in vitro maturation of guinea pig thymocytes (Lai et al.,
2002). Its action on macrophage function is well established: in rats, it accelerates clearance of colloidal carbon; in humans, in vivo and in vitro, it increases the metabolic activity of
blood monocytes and their affinity for the Fc fragment of IgG. Levamisole does not act directly on antibodies synthesis, but may enhance the responses to T dependent antigens by stimulation of T helper cells, even in normal, non immunosuppressed individuals. An interferon-like activity has been detected in serum of mice after parenteral inoculation of levamisole.
1.12 Some Plants With Immunological Potential
Plants species Part used Extract Model used
Hibiscusrosa
sinensis Flowers
Aerial
Hydro-alcoholic
Extract
Carbon clearance method, Cellular mediated immunity, Immunostimulatory
Carbon clearance method, Cellular mediated
Cleome gynandra
parts Ethanolic extract
Pe.Ether,
immunity, Immunostimulatory
Trikatu mega TriAmrit (Termi nalia, Allium, Tinospora) Nyctanthes
Aerial parts
Aerial parts
Benzene, Choloroform Pe.Ether, Benzene, Choloroform
Carbon clearance assay, delayed hypersensitivity test
Carbon clearance assay, delayed hypersensitivity test
Humoural immunity, delayed-type
arbortristis Leaf Ethanolic extract
hypersensitivity
Cissampelos
pareira Roots
Alkaloidal
fraction Humoural antibody titre
Bauhinia Vareigata Stem bark Acetone: water Human Neutrophils
Tinospora
Cordifolia Stems Ethanolic extract
Balanite Roxburghi Leaf Ethanolic extract
Ficus carica Leaf Ethanolic extract
Bone marrow cellularity and α‐Esterase cells, Zinc sulphate turbidity test
Carbon clearance test, serum immunoglobulin
Cellular immune response, Humoral antibody response
Capparis Zeylanica Leaf Alcoholic extract Phagocytosis; DTH
Trapa Bispinosa Fruits Aqueous extract Neutrophils, Haemagglutination titre. Aloe vera Leaves Saline extract Haematological, Serological studies. Heracleum
Persicum Fruits Aqueous Extract Haemagglutination titre, DTH
Tinospora
Cordifolia Stem Alcoholic extract Immunostimulant, macrophase chemotaxis
Ocimum sanctum
Whole
plant Aqueous extract
Stem
Enhance the production of RBC, WBC and haemoglobin
Bauhinia variegate
Chlorophytum
bark Ethanolic extract Neutrophil adhesion , Phagocytic activity
In Vivo Phagocytosis Using Carbon
Borivilianum Roots Ethanolic extract Methanolic
Clearance Method
Morus alba linn. Leaf
Extract Humoural immunity, serum immunoglobulin
Aesculus indica Leaf Petroleum ether Neutrophil index, Neutrophil AdhesionTable 1: some plants with immunological potential
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ELUCIDATION OF SOME IMMUNOLOGICAL AND BIOCHEMICAL NATURE OF THE LEAVES OF SENNA MIMOSOIDES
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