ELUCIDATION OF SOME IMMUNOLOGICAL AND BIOCHEMICAL NATURE OF THE LEAVES OF SENNA MIMOSOIDES

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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