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

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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 m g 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 levamisole 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 levamisole (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 elicited 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 levamisole respectively. The humoural antibody (HA) titre and DTH response compare well with that of levamisole, 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 decreased 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 levamisole 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 selenium showed the same trend. Histopathological studies showed that the extract and levamisole 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, 18.8 22, 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.

TABLE OF CONTENTS

Title Page

Certification

Dedication

Acknowledgement

Abstract

Table of Contents

List of Figures

List of Tables

List of Plates

List of Abbreviation

CHAPTER ONE: INTRODUCTION

1.1 Overview of the Human Immune System

1.2 The Cells of the Immune System

1.2.1 T-Lymphocytes

1.2.2 B-Lymphocytes

1.2.3 Natural Killer (Nk) Cells

1.2.4 Monocyte and Macrophages

1.2.5 Antigen-Presenting Cells (APCs)

1.2.6 Phagocytes

1.2.7 Neutrophils

1.2.8 Basophils and Mast Cells

1.2.9 Eosinophils

1.3 Innate (Nonspecific) Immunity

1.4 Adaptive Immunity

1.5 Humoural Immunity

1.6 Cell-Mediated Immunity (CMI)

1.7 Mediators of the Immune System

1.7.1 Cytokines

1.7.2 Complement System

1.8 Blood

1.9 The Concept of Immunomodulation

1.10 Cyclophosphamide (CP)

1.10.1 Metabolism of Cyclophosphamide

1.10.2 Mechanism of Action of Cyclophosphamide

1.11 Levamisole

1.12 Some Plants with Immunological Potential

1.13 The Liver and Its Function

1.14 The Overview of the Antioxidant Physiology of Human

1.15 Hepatotoxicity

1.16 Liver Histology

1.17 Biotransformation of Hepatotoxicants

1.18 Mechanism of Hepatotoxicity

1.19 Carbon tetrachloride (CCl4)

1.20 Lipid Peroxidation

1.21 Glutathione

1.21.1 Alanine Aminotransferases- the Standard Clinical Biomarker of Hepatotoxicity

1.21.2 Aspartate Aminotransferase (AST)

1.21.3 Alkaline Phosphatase (ALP)

1.21.4 Glutathione S-Transferase

1.22 Silymarin

1.23 Bilirubin

1.24 Serum/ Plasma Proteins

1.25 Phosphate

1.26 Calcium

1.27 Sodium

1.28 Potassium (K)

1.29 Magnesium

1.30 Zinc (Zn)

1.31 Selenium (Se

1.32 Iron

1.33 Breast Milk Toxicity

1.34 Disaccharides

1.35 Botanical Outline of Senna Mimosoides

1.36 Previous Investigation Carried Out on Senna Species

1.37 Aim of the Study

1.40 Specific Research Objectives

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials

2.1.1 Plant Material

2.1.2 Animal Material

2.1.3 Chemicals and Reagents

2.1.3.1Chemicals

2.1.3.2Reagents

2.2 Methods

2.2.1 Aqueous Extraction

2.2.2 Experimental Design

2.2.3 Phytochemical Analysis

2.2.3.1 Qualitative Phytochemical Analysis

2.2.3.1.1 Test for Saponins

2.2.3.1.2 Test for Alkaloids

2.2.3.1.3 Test for Tannins

2.2.3.1.4 Test for Flavonoides

2.2.3.1.5 Test for Terpenoids

2.2.3.1.6 Test for Steroids

2.2.3.1.7 Test for Phenols

2.2.3.1.8 Test for Glycosides

2.2.3.1.9 Test for Reducing Sugar

2.2.3.1.10 Test for Cyanide

2.2.3.1.11 Test for Soluble Carbohydrate (Molisch Test)

2.2.3.2 Quantitative Phytochemical Analysis

2.2.3.2.1 Test for Saponins

2.2.3.2.2 Test for Alkaloids

2.2.3.2.3 Test for Tannins

2.2.3.2.4 Test for Flavonoids

2.2.3.2.5 Test for Terpenoids

2.2.3.2.6 Test for Steroids

2.2.3.2.7 Test for Glycosides

2.2.3.2.8 Test for Reducing Sugar

2.2.3.2.9 Test for Soluble Carbohydrate

2.2.3.2.10 Test for Cyanide

2.2.3.2.11 Test for Phenols

2.2.4 Determination of Biological Activity

2.2.4.1 Acute Toxicity and Lethality

2.2.4.2 Immunomodulatory Activity of Extracts

2.2.4.2.1 Preparation of Antigen

2.2.4.2.2 Delayed Type Hypersensitivity (DTH) Reaction

2.2.4.2.3 Humoural Antibody (HA) Synthesis

2.2.4.3 Cyclophosphamide-Induced Myelosuppression

2.2.4.4 Determination of Haematological Parameter

2.2.4.4.1 Determination of WBC Count

2.2.4.4.2 Determination of PCV Concentration

2.2.4.4.3 Determination of Hb Concentration

2.2.4.4.4 Determination of RBC Count

2.2.4.5 Effect of the Extract on in vivo Leukocyte Mobilization

2.2.4.6 Determination of the Effect of the Extract on CCl4 Induced Hepatotoxicity

2.2.4.6.1 Assay of Serum Alanine Aminotransferase (ALT) Activity

2.2.4.6.2 Assay of Aspartate Aminotransferase (AST) Activity

2.2.4.6.3 Assay of Serum Alkaline Phosphatase (ALP) Activity

2.2.4.6.4 Determination of Serum Bilirubin

2.2.4.6.5 Determination of Catalase Activity

2.2.4.6.6 Determination of Reduced Glutathione Level

2.2.4.6.7 Determination of Superoxide Dismutase (SOD) Activity

2.2.4.6.8 Assay of Glutathione S-Transferase (GST) Activity

2.2.4.6.9 Assay of Glutathione Peroxidase Activity

2.2.4.6.10 Determination of Malondialdehyde (MDA) Concentration

2.2.4.7 Serum Inorganic Ion Determination

2.2.4.7.1 Determination of Serum Iron Concentration

2.2.4.7.2 Determination of Serum Selenium Concentration

2.2.4.7.3 Determination of Serum Zinc Concentration

2.2.4.7.4 Determination of Serum Calcium Concentration

2.2.4.7.5 Determination of Serum Phosphate Concentration

2.2.4.7.6 Determination of Serum Magnesium Concentration

2.2.4.7.7 Determination of Serum Sodium and Potassium

2.2.4.8 Determination of Serum Protein Concentration

2.2.4.9 Histopathological Study

2.2.4.10 Determination of the Effect of the Extract on the Activity of Lactase 65

2.2.5 Statistical Analysis

CHAPTER THREE: RESULTS

3.1 Extract of Senna mimosoides

3.2 Phytochemical Composition of Aqueous Extract of S. mimosoides Leaves

3.2.1 Qualitative Phytochemical Composition of Aqueous Extract of S. mimosoides Leaves

3.2.2 Qualitative Phytochemical Composition of Aqueous Extract of S. mimosoides Leaves

3.3 LD50

3.4 Effect of Aqueous Extract of S. mimosoides Leaves on In Vivo Leukocyte Migration

3.5 Effect of Aqueous Extract of S. mimosoides Leaves on Humoural Immunity

3.6 Effect of Aqueous Extract of S. mimosoides Leaves on Cell Mediated Immunity

3.7 Effect of Aqueous Extract of S. mimosoides Leaves on Serum PCV Level

3.8 Effect of Aqueous Extract of S. mimosoides Leaves on Serum Hb Concentration

3.9 Effect of Aqueous Extract of S. mimosoides Leaves on Serum WBC Level

3.10 Effect of Aqueous Extract of S. mimosoides Leaves on Serum Red Blood Cell Level

3.11 Effect of Aqueous Extract of S. mimosoides Leaves on MCH Concentration Level

3.12 Effect of Aqueous Extract of S. mimosoides Leaves on Mean Cellular Volume

3.13 Effect of Aqueous Extract of S. mimosoides Leaves on Serum MCH Level

3.14 Effect of Aqueous Extract of S. mimosoides on Serum Level of AST

3.15 Effect of Aqueous Extract of S. mimosoides on Serum Level of Alanine Transaminase

3.16 Effect of Aqueous Extract of S. mimosoides on Serum Level of Alkaline Phosphatase

3.17 Effect of Aqueous Extract of S. mimosoides on Serum Level of Bilirubin

3.18 Effect of Aqueous Extract of S. mimosoides on the Activity of Glutathione Peroxidase

3.19 Effect of Aqueous Extract of S. mimosoides on the Activity of Superoxide Dismutase

3.20 Effect of Aqueous Extract of S. mimosoides on the Activity of Catalase

3.21 Effect of Aqueous Extract of S. mimosoides on Lipid Peroxidation

3.22 Effect of Aqueous Extract of S. mimosoides on the Activity of Glutathione S-transferase

3.23 Effect of Aqueous Extract of S. mimosoides on Glutathione Level

3.24 Effect of Aqueous Extract of S. mimosoides on Total Serum Protein

3.25 Effect of Aqueous Extract of S. mimosoides on Serum Level of Sodium

3.26 Effect of Aqueous Extract of S. mimosoides on Serum Level of Magnesium

3.27 Effect of Aqueous Extract of S. mimosoides on Serum Level of Iron

3.28 Effect of Aqueous Extract of S. mimosoides on Serum Level of Potassium

3.29 Effect of Aqueous Extract of S. mimosoides on Serum Level of Phosphate

3.30 Effect of Aqueous Extract of S. mimosoides on Serum Level of Calcium

3.31 Effect of Aqueous Extract of S. mimosoides on Serum Level of Zinc

3.32 Effect of Aqueous Extract of S. mimosoides on Serum Level of Selenium

3.33 Effect of Aqueous Extract of S. mimosoides on the Activity of Lactase

3.34 Histopathological Examination on the Liver Control Group A

3.35 Histopathological Examination of Liver Cells of Rats in Group B

3.36 Histopathological Examination of Liver Cells of Rats in Group C

3.37 Histopathological Examination of Liver Cells of Rats in Group D

3.38 Histopathological Examination of Liver Cells of Rats in Group E

3.39 Histopathological Examination of Liver Cells of Rats in Group F

CHAPTER FOUR: DISCUSSION

4.1 Discussion

4.2 Conclusion

4.3 Suggestions for Further Studies

References

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 important 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, immediately after delivery, breast milk is usually dropped on the leaves of cocoyam or on ants to check its toxicity.Toxic breast milk 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. mimosoides was 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.

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.

1.2 The 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-lymphocytes perform the actual destruction of the invading microorganism (Lukashenka et 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).

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 gamma globulins) (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 opsonization. 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 Thiel, 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 controlling 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.

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 serine proteases and antimicrobial peptides such as cathelicidin (Brinkmann et al., 2004).

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 leukotrienes 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).

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.

1.3 Innate (Nonspecific) Immunity

Innate or nonspecific 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-Paschoalotto et al., 2011).

1.4 Adaptive Immunity

Adaptive (acquired, specific) immunity is capable of recognizing and selectively eliminating foreign microorganism and molecules. These host defences are mediated by two interrelated 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’.

1.5 Humoural 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).

1.6 Cell-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.

1.7 Mediators 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.

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 hemoglobin 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 hematological 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.

1.9 The 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.

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 Jonge 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.

Metabolism of Cyclophosphamide

Cyclophosphamide is activated by hepatic microsomal mixed function oxidases cytochrome P450 to form 4–hydroxycyclophosphamide (4-OHCP), wh ich 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.

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 (Choong et 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 co-oxidation 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 (Zhang et al., 2006). Golgi complexes will be stimulated. The rough endoplasmic reticulum will be focally degranulated, while the smooth endoplasmic reticulum appears considerably proliferated.

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.

1.13 The Liver and its Function

Liver, the most important and largest organ of human being regulates various physiological processes in the body such as biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction (Ahsan et al., 2009 and Sarkar et al., 2005). The major functions of the liver is carbohydrate, protein and fat metabolism; detoxification; secretion of bile; synthesis of glucose from glycogenesis; conversion of sugar glucose into glycogen and storing it until the body needs it; storage of vitamin and minerals (Ahsan, et al., 2009). Liver cells produce plasma proteins, clotting factors, urea and lipids or fatty substances that include triglycerides, cholesterol and lipoproteins. The liver makes bile acids that breaks down the fat in food; are necessary for the absorbtion of vitamins A, D and E, by the body; remove toxic substances; serves as a filter that separates out harmful substances from the bloodstream and excretes them (Saukkonen et al., 2006). Smooth endoplasmic reticulum of the liver is the principal ‘metabolic clearing house’ for both endogenous chemicals like cholesterol, steroid hormones, fatty acids, proteins, and exogenous substances like drugs and alcohol (Singh et al., 2011). Generally, it metabolizes all foreign compounds by making a non-polar molecule more polar so that it can either be excreted into the urine via the kidney or be secreted into the feaces through bile. The central role played by liver in the clearance and transformation of chemicals exposes it to toxic injury (Sarkar et al., 2005). It also screens out bilirubin, a reddish-yellow pigment formed by the breakdown of haemoglobin in worn-out red blood cells by combining it with bile and passing it the duodenum to be excreted. Inability of the liver to screen out bilirubin leads to jaundice, characterised by development of a yellowish colour in the whites of the eyes and in the skin (Singh et al., 2011). The liver produces albumin, a protein found in blood and cholesterol that is critical to the make-up of the outer membrane of cells.

Continuous exposure of the liver to environmental toxins, abuse by poor drug habits and excessive alcohol can lead to various ailments like hepatitis, cirrhosis and alcoholic liver disease (Ahsan et al., 2009; Sharma et al., 1991; Subramonium and Pushpangadan, 1999). Liver damage is associated with oxidative stress, cellular necrosis, increase in lipid peroxidation, depletion in glutathione (GSH) level, elevation in serum levels of many biochemical markers like aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglycerides, cholesterol, bilirubin, and lactate dehydrogenase (LDH) (Singh et al., 2011). Despite the frequent occurrence of hepatic diseases, its morbidity and high mortality, its medical management is currently inadequate, so far not yet any therapy has successfully prevented the progression of hepatic disease, even though newly developed drugs have been used to treat chronic liver disorders, these drugs have often side effects. This has led to recent researches involving the use of suitable herbal drugs that could replace the chemical ones.

1.14 The Overview of the Antioxidant Physiology of Humans

The ability to utilize oxygen has provided humans with the benefit of metabolizing fats, proteins, and carbohydrates for energy. Oxygen is a highly reactive atom that is capable of becoming part of potentially damaging molecules commonly called “free radicals.” Free radicals are capable of attacking the healthy cells of the body, causing them to lose their structure and function.Reactive oxygen species (ROS) is a term which encompasses all highly reactive, oxygen-containing molecules, including free radicals. Types of ROS include the hydroxyl radical, the superoxide anion radical, hydrogen peroxide, singlet oxygen, nitric oxide radical,hypochlorite radical, and various lipid peroxides (Percival, 1998). All are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes,and other small molecules, resulting in cellular damage.ROS are generated by a number of pathways. Most of the oxidants produced by cells occur as:

• A consequence of normal aerobic metabolism.

• Oxidative burst from phagocytes (white blood cell s).

• Xenobiotic metabolism, i.e., detoxification of to xic substances.

To protect the cells and organ systems of the body against reactive oxygen species, humans have evolved a highly sophisticated and complex antioxidant protection system. It involves a variety of components, both endogenous and exogenous in origin, that function interactively and synergistically to neutralize free radicals (Percival, 1998).

These components include:

• Nutrient-derived antioxidants like ascorbic acid (vitamin C),tocopherols and tocotrienols (vitamin E), carotenoids, and otherlow molecular weight compounds such as glutathione and lipoicacid.

• Antioxidant enzymes, e.g., superoxide dismutase, glutathioneperoxidase, and glutathione reductase, which catalyze freeradical quenching reactions.

• Metal binding proteins, such as ferritin, lactofe rrin, albumin, andceruloplasmin that sequester free iron and copper ions that arecapable of catalyzing oxidative reactions.

• Phytonutrients present in a widevariety of plant foods e.g.flavonoids, flavones, flavonols, andproanthocyanidins.

Hydroxyl radical is neutralized by vitamin C, glutathione,flavonoids, lipoic acid; Superoxide radical by vitamin C, glutathione,flavonoids, SOD; Hydrogen peroxide by vitamin C, glutathione, betacarotene, vitamin E, CoQ10,flavonoids, lipoic acid; Lipid peroxides by beta carotene, vitamin E,ubiquinone, flavonoids,glutathione peroxidase (Percival, 1998).

Vitamin C, E and beta carotene are among the most widely studied dietary antioxidants. Vitamin C being water-soluble neutralizes ROS in the aqueous phase before lipid peroxidation is initiated. Vitamin E, a lipid-soluble antioxidant,is the most effective chain-breaking antioxidant within the cell membrane.Beta carotene and other carotenoids are also believed to provide antioxidant protection to lipid-rich tissues.

In addition to dietary antioxidants, the body also relies on several endogenous defense mechanisms to help protect against free radical-induced cell damage. Endogenous antioxidants include the following: glutathione peroxidase, copper/zinc and manganese-dependent superoxide dismutase (SOD),iron-dependent catalase,selenium-dependent glutathione peroxidase,bilirubin, glutathione, lipoic acid, N-acetylcysteine, NADPH, NADH, Ubiquinone (coenzyme Q10), Uric acid.Albumin (copper), ceruloplasmin (copper), metallothionein (copper), ferritin (iron), myoglobin (iron), transferrin (iron) are endogenous protein that help in protecting the body from free radicals (Percival, 1998).

1.15 Hepatotoxicity

Hepatotoxicity refers to liver dysfunction or liver damage that is associated with an overload of drugs or xenobiotics (Singh et al., 2011). Hepatotoxicity can be caused by a wide variety of pharmaceutical agents, natural products, chemicals or environmental pollutants and dietary constituents. Hepatotoxicity is associated with lipid peroxidation, enzymatic leakage of alanine aminotransaminase, alkaline phosphatase and depletion of reduced glutathione and total thiols (Bai, 2011; Appiah et al., 2009). There is a commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes (Palanisamy et al., 2007; Palani, 2009). Modern medicine has little to offer for elevation of hepatic diseases and it is chiefly the plant based preparations that are employed for the treatment of liver disorders. The drugs offered by medicine for the treatment of liver diseases are corticosteroids and immunosuppressants which provide only symptomatic relief mostly without influencing the disease process and their use is associated with the risk of relapse and danger of side effects (Mukherjee and Mukherjee, 2009).

Hepatotoxicity may result not only from direct toxicity of the primary compound but also from a reactive metabolite or from an immunologically-mediated response affecting hepatocytes, biliary epithelial cells and/or liver vasculature (Saukkonen et al., 2006; Deng et al., 2009). The hepatotoxic response elicited by a chemical agent depends on the concentration of the toxicant which may be either parent compound or toxic metabolite, differential expression of enzymes and concentration gradient of cofactors in blood across the acinus (Singh et al., 2011). Hepatotoxicity related symptoms may include jaundice, pruritus, severe abdominal pain, nausea or vomiting, weakness, severe fatigue, continuous bleeding, skin rashes, generalized itching, swelling of the feet and/or legs, abnormal and rapid weight gain in a short period of time, dark urine and light colored stool (Bleibel et al., 2007). However, severe intoxication with hepatotoxic agents can lead to liver necrosis and death of the organism if left untreated.

The hepatotoxic effects of chemical agents may involve different mechanisms of cyto lethality. These mechanisms may have either direct effect on organelles like mitochondria, endoplasmic reticulum, the cytoskeleton, microtubules and nucleus or indirect effect on cellular organelles through the activation and inhibition of signalling kinases, transcription factors and gene-expression profiles (singh et al., 2011). The resultant intracellular stress may lead to cell death caused by either cell shrinkage and nuclear disassembly (apoptosis) or swelling and lysis (necrosis).

1.16 Liver Histology

Liver histology or electron microscopy gives precious information regarding the mitochondrial origin of Drug Induced Liver Injury for they can reveal not only ultrastructural changes in liver mitochondria (enlargement or swelling, disruption of cristae), but also the presence of small lipid droplets within the cytoplasm suggesting microvesicular steatosis (Labbea et al., 2008). The presence of microvesicular steatosis is highly suggestive of a strong inhibition of mitochondrial FAO (Labbea et al., 2008). It is noteworthy, however, that pure microvesicular steatosis is rare, and that this lesion is often accompanied by macro vacuolar steatosis. The presence of steatohepatitis can also suggest drug-induced mitochondrial dysfunction. Moreover, ultrastructural alterations of mitochondria are not necessarily a direct consequence of drug toxicity. However, investigations should be performed to test the possibility that this liver lesion could be due also (or instead) to an increased hepatic de novo lipogenesis, such as that triggered by insulin resistance in a context of body fatness (Pessayre and Fromenty, 2005; Begriche et al., 2006). It is important to mention that drugs can indirectly impair mitochondrial FAO in liver and cause steatosis (or steatohepatitis) by inducing lipoatrophy (i.e. a reduction in body fat mass) (Igoudjil et al., 2007). Fatty liver (or steatohepatitis) secondary to lipoatrophy is, at least in part, the consequence of a lower production of leptin by the white and necrosis in liver does not specifically imply direct toxicity of the drug on mitochondria.

1.17 Biotransformation of Hepatotoxicants

Liver plays a central role in biotransformation and disposition of xenobiotics. Metabolism of exogenous compounds can modulate the properties of hepatotoxicity by either increasing its toxicity (intoxication or metabolic activation) or decreasing its toxicity (detoxification) (Singh et al., 2011). Most of the foreign substances are lipophilic thus enabling them to cross the membranes of intestinal cells. They are rendered more hydrophilic by biochemical processes in the hepatocyte, yielding water-soluble products that are exported into plasma or bile by transport proteins located on the hepatocyte membrane and subsequently excreted by the kidney or gastrointestinal tract (Tostmann et al., 2008). The hepatic biotransformation involves Phase I and Phase II reactions. Phase I involves oxidative, reductive, dehydroxylation and demethylation pathways, primarily by way of the cytochrome P-450 enzyme system located in the endoplasmic reticulum, which is the most important family of metabolizing enzymes in the liver. Endoplasmic reticulum also contains a NADPH-dependent mixed function oxidase system, the flavin-containing monooxygenases, which oxidizes amines and sulphur compounds. Phase I reactions often produce toxic intermediates which are rendered non-toxic by phase II reactions. Phase II reactions involve the conjugation of chemicals with hydrophilic moieties such as glucuronide, sulfate or amino acids and lead to the formation of more water-soluble metabolite which can be excreted easily (Singh et al., 2011). Another Phase II reaction involves glutathione which can covalently bind to toxic intermediates by glutathione s-transferase (Singh et al., 2011). As a result, these reactions are usually considered detoxification pathways. However, this phase can also lead to the formation of unstable precursors to reactive species that can cause hepatotoxicity.

Many substances can influence the cytochrome P450 enzyme mechanism. Such substances can serve either as inhibitors or inducers. Hepatotoxicity may also arise from an adaptive immune response to proteins bound to the hepatotoxicity or its metabolites (Park et al., 2001; Ju and Uetrecht, 2002). Random exposure to lipopolysaccharides (LPS) or other inflammatory conditions could potentiate hepatotoxicity by involving a combination of fibrin deposit induced hypoxia and neutrophil-mediated cell damage (Luyendyk et 6al., 2005).

Many hepatotoxicants e.g. carbon tetrachloride, amodiaquine, acetaminophen, halothane, isoniazid, allyl alcohol and bromobenzene are metabolized to chemically reactive toxic metabolites which can covalently bind to crucial cellular macromolecules thus disrupting critical cellular functions (Singh et al., 2011). The reactive metabolites may also alter liver proteins leading to an immune response and immune-mediated injury. Most hepatotoxicants causes hepatotoxicity through lipid peroxidation and redox cycling which leads to cell death due to oxidative stress which arises when there is alteration in the ratio of intracellular pro-oxidant to antioxidant in favor of prooxidants (Singh et al., 2011). Lipid peroxy radicals lead to increased cell membrane permeability, decreased cell membrane fluidity, inactivation of membrane proteins and loss of polarity of mitochondrial membranes. Metal ions like iron and copper participate in redox cycling while cycling of oxidised and reduced forms of a toxicant leads to the formation of reactive oxygen free radicals which can deplete glutathione through oxidation or oxidize critical protein sulfhydryl groups involved in cellular or enzymatic regulation or can initiate lipid peroxidation (Singh et al., 2011).

1.18 Mechanism of Hepatotoxicity

The liver has a remarkable ability to regenerate itself following injury or inflammation and it has nutrient reserves it can tap when it is damaged (Sharma and Biyani, 2012). There are two stages of liver disease. The first stage known as compensated liver disease is characterised by inflammation which is followed by fibrosis or scar tissue which advances to cirrhosis and then finally the liver becomes cancerous. The lobules and portal triad (the area where the large portal vein and its branches enter the liver) are part of the liver that is prone to inflammation and scarring in the early stage of liver disease (Sharma and Biyani, 2012). In this type of disease, the injury can initially be tolerated and resisted, due to the liver’s ability to regenerate and compensate for the damage and in this case, the liver still continues with its functions. In the second stage known as decompensated liver disease, the fibrosis tissue or scarring expands and “bridges” between portal areas . Moreover, the scarring is so extensive that it expands to the central area of the liver and the liver changes its shape or “architecture” due to the scarring and tissue regeneration attempts. This is the end stage of liver disease for in this stage, the liver loses its ability to regenerate liver tissue and its filtering and nutrient storing abilities are damaged by scar tissue because the liver cannot compensate for the ongoing damage (Sharma and Biyani, 2012).

Main patterns of liver injury during hepatotoxicity may include zonal necrosis, hepatitis, cholestasis, steatosis, granuloma, vascular lesions, neoplasm and veno-occlusive diseases (Singh et al., 2011). A liver contains hepatocytic or liver cells, a porous lining, tissue macrophages called Kupffer cells and stellate cells (formerly called lto cells or lipocytes or fat-storing cells). The stellate cells are fairly inactive cells that store vitamin A and maintain the livers membrane around different liver sections. Injury to the adjacent membrane or kupffer cell activates the stellate cells which begin to proliferate shedding their vitamin A and reconstituting themselves to produce fibrous material or scar tissue. The scar tissue cells accumulate and hinder liver functions.

1.19 Carbon Tetrachloride (CCl4)

CCl4 is a colourless liquid, non flammable, and is heavier than air.CCl4 is well absorbed from the gastrointestinal and respiratory tract in animals and humans.In humans, acute symptoms after CCl4 exposure are independent of the route of intake and are characterized by gastrointestinal and neurological symptoms, such as nausea, vomiting, headache, dizziness, dyspnoea and death. Liver damage appears after 24 hr or more. Kidney damage is evident often only 2 to 3 weeks following the poisoning.CCl4 treatment of female mice resulted in marked suppression of both humoural and cell-mediated immune functions.

On entry into the body, CCl4 causes a lot of injury to the organs of the body including the lungs, heart, gastrointestinal tract, kidneys, CNS, and liver (Etim et al., 2008). Common synonyms of CCl4 are Carbon, carbon chloride, tetrachloromethane, carbon tet, methane tetrachloride, perchloroethylene, tetrachlorocarbon.

Fig. 2: Biotransformation of CCl4

(From Egbuna et al., 2011)

CCl4 is metabolically activated by the cytochrome P450 -dependent mixed oxidase present in the endoplasmic reticulum. The first step is the biotransformation of CCl4 initiated by cytochrome P450 Mediated transfer of an electron to the C-l bond, forming an anion radical that eliminates chloride, thus forming the reactive trichloromethyl radical (Egbuna et al., 2011).The most important pathway in the elimination of trichloromethyl radicals is the reaction with molecular oxygen, resulting in the formation of trichloromethyl peroxyl radicals (CCl 3 OOC), which can react further to form phosgene.This intermediate, being more reactive than trichloromethyl radical, interacts with lipids, causing lipid peroxidation and production of 4-hydroxy alkenals (Percival, 1998). Condensation of phosgene with cysteine leads to the formation of 2-oxothiazolidine-4-carboxylic acid.The condensation of phosgene with glutathione (GSH), results in the formation of glutathionyl dithiocarbonate.Phosgene may be detoxified by reaction with water to produce carbon dioxide or with glutathione or cysteine.

CCl3 in the presence of oxygen generated by metabolic leakage from mitochondria reacts with sulfhydryl group, protein-thiol and reduced Glutathione (GSH), thus covalently bind with the cell membrane and leads to membrane lipid peroxidation which ends at necrosis of the cell (Lee et al., 2007). This is followed by chloromethylation, saturation, peroxidation and progressive destruction of the unsaturated fatty acid of the endoplasmic reticulum membrane phospholipids. These result in changes of structure of the lipids of endoplasmic reticulum rich in polyunsaturated fatty acids and other membrane, loss of metabolic enzyme activation, reduction of protein synthesis and loss of glucose-6 phosphatase activation leading to liver damage (degeneration, necrosis and fibrosis of liver cell) (Galisteo et al., 2000; Ahsan et al., 2009). The hepatotoxicity involves 2 phases. The initial phase involves metabolism of CCl4 by cytochrome P450, which leads to the formation of free radicals and lipid peroxides. The second step involves activation of Kupffer cells, probably by free radicals. Activation of Kupffer cells is accompanied by production of proinflammatory mediators.

1.20 Lipid Peroxidation

Lipid peroxidation is the oxidative degradation of polyunsaturated fatty acids by free radicals leading to membrane damage. Free radicals induce lipid peroxidation in polyunsaturated lipid rich areas like brain and liver (Selvam et al., 2010). Lipid peroxidation is a well-defined mechanism of cellular damage in animals and plants. Oxidative modification of lipids can be induced in vitro by a wide array of pro-oxidant agents and occurs in vivo during aging and under certain pathologic conditions (Kim et al., 2011). Lipid peroxides are indicators of cellular oxidative stress that decompose to form more complex and reactive compounds such as MDA and 4-hydroxynonenal. These aldehydic secondary byproducts of lipid peroxidation are generally accepted markers of oxidative stress. H2O2is eliminated by various antioxidant enzymes such as CAT and GPX which convert H2O2 into water. Toxic O2-, H2O2, and OH radicals are efficiently eliminated by non-enzymatic (α-tocopherol, б-carotene, phenolic compounds, ascorbate, glutathione) and enzymatic antioxidants (Kim et al., 2011).

At several upstream sites of the MRC (most probably complexes I and III), a small fraction of electrons provided by FADH2 or NADH can directly react with oxygen, to generate the superoxide anion radical (Labbea et al., 2008). This radical is then dismutated by the mitochondrial manganese superoxide dismutase (MnSOD) into hydrogen peroxide (H2O2), which is detoxified into water by the mitochondrial glutathione peroxidase (Labbea et al., 2008). Hence, in the normal (non-diseased) state, most of the ROS generated by the MRC is detoxified by the mitochondrial antioxidant defences. However, this detoxification process can be overwhelmed in some circumstances. One such circumstance is the depletion of mitochondrial, reduced glutathione. Normally, glutathione peroxidase plays a key role in H2O2 detoxification, as liver mitochondria do not have catalase (Labbea et al., 2008). However, glutathione peroxidase needs adequate amounts of reduced glutathione in the mitochondrial matrix to detoxify H2O2. The depletion of mitochondrial glutathione below a critical threshold can impair mitochondrial H2O2 detoxification, which can trigger mitochondrial dysfunction, mitochondrial permeability transition (MPT) and cell death (Fernandez-Checa et al., 2005). Mitochondrial antioxidant enzymes can also be overwhelmed when electron transfer within the MRC is chronically hindered. A partial block in the flow of electrons within the MRC leads to their accumulation within MRC complexes. This oxidative damage aggravates mitochondrial dysfunction to further augment electron leakage and ROS formation, thus leading to a vicious circle (Pessayre et al., 2007; Igoudjil et al., 2006).

The Mitochondrial Permeability Transition (MPT) pore located at contact sites between the outer and inner mitochondrial membranes is composed of proteins like the voltage-dependent anion channel (VDAC) located in the outer membrane, the adenine nucleotide translocator (ANT) (also called ADP/ATP translocase) in the inner mitochondrial membrane and cyclophilin D in the mitochondrial matrix (Labbea et al., 2008). MPT pores are kept closed, but in certain conditions they rapidly open, increasing the permeability of the mitochondrial membranes to compounds which their molecular weight is <1.5 kDa. This leads to collapse of transmembrane potential (Dwm) and expansion of the mitochondrial matrix due to accumulation of water (Labbea et al., 2008). Consequently, the outer membrane ruptures releasing pro-apoptotic proteins from the intermembrane space into the cytosol. The release of apoptosis-inducing factor (AIF) triggers the fragmentation of large-sized nuclear DNA. The release of a protein called ‘second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO)’ inactivates the inhibitor of apoptosis proteins (IAPs). Finally, the released cytochrome c binds in the cytosol to Apaf-1 in an ATP-dependent manner to activate caspase-9. The leakage of cytochrome c from the mitochondria eventually impairs electron transfer within the MRC and enhances ROS generation. Mitochondrial permeability transition can cause either apoptosis or necrosis. MPT pore opening, is calcium-dependent and can be triggered by a variety of endogenous compounds such as iron, ROS, nitric oxide, free fatty acids (and acyl-CoA derivatives), ceramide, bile salts, drugs and extracellular cytokines such as tumour necrosis factor-a (TNF-a) and Fas ligand (Fas-L) acting through their plasma membrane receptors (Labbea et al., 2008). Some drugs cause MPT by inducing the release of pro-apoptotic proteins from mitochondria through indirect mechanisms. For instance, troglitazone and acetaminophen activate c-Jun N-terminal protein kinase (JNK), thus inducing the cleavage of Bid, the translocation of this pro-apoptotic protein to the outer mitochondrial membranes and the release of cytochrome c from mitochondria (Gunawan et al., 2006). Another mechanism is seen during the extensive formation of reactive metabolites, which increases cytosolic calcium that then enters into the mitochondria to trigger MPT and apoptosis (Haouzi et al., 2000). 1.21 Glutathione

Glutathione, a tripeptide containing a sulfhydryl group, is a highly distinctive amino acid derivative and one of the most important non-enzymatic antioxidant. It is a nucleophilic thiol, a strong reductant and a critical determinant of tissue susceptibility to oxidative damage (Labib et al., 2001). GSH can react with electrophiles and ROS thus protecting thiol groups of proteins from oxidation. It also serves as a substrate for GPx and glutathione s-transferase. The depletion of GSH in the blood may be explained by increased utilization of GSH for removal of ROS and lipid damaged products or due to impairment of synthesis (Labib et al., 2001).

The GSH antioxidant system consists of an array of non-enzymic and enzymic reaction pathways involving the neutralization of free radical species. GSH acts synergistically with vitamin-E in inhibiting oxidative stress and acts against lipid peroxidation (Pradeep et al., 2007). GPx utilizes it for the decomposition of lipid hydroperoxides and other reactive oxygen species (ROS) and glutathione s-transferase (GST) maximizes the conjugation of free radicals and various lipid hydroperoxides to GSH to form water-soluble products that can be easily excreted out (Shahjahan et al., 2004). The multiple mechanisms whereby enzymes are regulated include increased transcription and post-transcriptional modulation, which are apparently mediated through generation of reactive oxygen species and reduced glutathione (GSH) conjugate formation, respectively. Reduced glutathione acts as an antioxidant both intracellularly and extracellularly in conjunction with various enzymatic processes that reduce hydrogen peroxide and hydroperoxides by oxidizing reduced glutathione to its oxidized form and other mixed disulfides (Dahiru and Obidoa, 2007).

1.21.1 Alanine Aminotransferases- the Standard Clinical Biomarker of Hepatotoxicity

Alanine aminotransferase (ALT) a cytoplasmic liver enzyme present in hepatic and biliary cells is the most frequently relied biomarker of hepatotoxicity (Jensen et al., 2004) which plays an important role in amino acid metabolism and gluconeogenesis. Plasma concentrations increase with hepatocellular damage/necrosis, hepatocyte proliferation, or hepatocellular degeneration. It catalyzes the reductive transfer of an amino group from alanine to α-ketoglutarate to yield glutamate and pyruvate. This enzyme is usually released from the hepatocytes and leaks into circulation causing increase in its serum levels under hepatocellular injury or inflammation of the biliary tract cells resulting predominantly in an elevation of the alkaline phosphatase levels (Jensen et al., 2004). The estimation of this enzyme is a more specific test for detecting liver abnormalities since it is primarily found in the liver (Singh et al., 2011). However, lower enzymatic activities are also found in skeletal muscles and heart tissue.The extent of the enzyme change is related to the nature, closeness to toxic agent and duration of toxicity (Shi et al., 2003; Song et al., 2003). In an inflammatory condition, there is a leakage of cytoplasmic enzymes into circulation, hence ALT levels increased above that of AST.

1.21.2 Aspartate Aminotransferase(AST):

AST present in many tissues is useful in evaluating liver damage in small and large animals. It is another liver enzyme that aids in producing proteins by catalysing the reductive transfer of an amino group from aspartate to α-ketoglutarate to yield oxaloacetate and glutamate (Singh et al., 2011). It is not liver specific in any domestic animal species for it is also found in other organs like heart, muscle brain and kidney. AST is present in both the cytoplasm and mitochondria of hepatocytes (and many other cells) and will elevate in states of altered membrane permeability. In such cases, levels are expected to be less than in states of frank necrosis, when both cytoplasmic and mitochondrial enzymes are released.

The magnitude of both AST and ALT elevations in serum is generally related to the number of hepatocytes affected. However, the level cannot be used to predict either the type of lesion or whether cell damage is reversible (leakage) or irreversible (frank necrosis) (kings and Perry, 2001). In fact, focal necrosis may yield a lower concentration of both AST and ALT than would severe, transient hypoxia in which all cells may be affected resulting in a potentially reversible alteration in membrane permeability and diffuse enzyme leakage. Equally increases in ALT and AST may be relatively mild in cases of severe cirrhosis/fibrosis of the liver since there is no ongoing hepatocellular damage. Another factor to be considered when interpreting AST and ALT levels is the rate of clearance from plasma. Both enzymes are molecularly too large to permit glomerular filtration and are primarily stereochemically denatured. The half-life of these enzymes is approximately 2-4 days and some prognostic information may be gleaned with this knowledge. Thus, if an elevated serum level falls by 50% after 2-4 days, the prognosis is generally more favourable than if the enzymes remain persistently elevated or are only slightly decreased after this time period.The ratio of serum AST to ALT can be used to differentiate liver damage from other organ damage.

1.21.3 Alkaline Phosphatase (ALP)

Alkaline phosphatases are a group of enzymes which catalyse the hydrolysis of a phosphate group from an organic molecule at an alkaline pH. They are called isoenzymes because they catalyse the same reaction in the same species but have different biochemical properties. ALP is primarily bound to cell membranes in the biliary ducts of the liver, intestines, kidney, placenta and bone. The liver isoenzyme will be elevated in any acute liver disease. In acute hepatocellular necrosis, ALT and AST are markedly elevated while ALP is only minimally elevated (Hyder et al., 2013). Intrahepatic and extrahepatic biliary obstruction causes more dramatic elevations of ALP, which in some cases can be 10-20 times the normal level. This is due to recycling as well as increased synthesis of the liver isoenzymes. Extrahepatic biliary obstruction can be caused if the hepatic or common bile duct is obstructed either partially or completely. Possible causes include tumour, granulomatus inflammation, abscesses, pancreatitis and duodenitis. It is elevated if bile excretion is inhibited by liver damage.

Increase in alkaline phosphatase and/or bilirubin with little or no increase in ALT is primarily a biomarker of hepatobiliary effects and cholestasis (Singh et al., 2011). In humans, increased ALP levels have been associated with drug induced cholestasis.

1.21.4 GlutathioneS-Transferase

GlutathioneS-transferase (GST) is an inducible phase II detoxification enzymes that catalyze the conjugation of glutathione with reactive metabolites formed during phase I of metabolism (Thapa and Walia, 2007). It is usually a nucleophilic attack by reduced glutathione (GSH) on nonpolar compounds that contain an electrophilic carbon, nitrogen, or sulphur atom. Their substrates include halogen nitrobenzenes, arene oxides, quinones, and α, β-unsaturated carbonyls (1–5) they metabolize cancer chemotherapeutic agents, insecticides, herbicides, carcinogens, and by-products of oxidative stress. Induction of GST synthesis is a protective mechanism that occurs in response to xenobiotic exposure. It is released quickly and in large quantities into the bloodstream during hepatocellular injury and the elevations in its activity are more rapid than AST or ALT.

Glutathione transferases catalyze the first of four steps required for the synthesis of mercapturic acids (Hayes et al., 2005). Subsequent reactions in this pathway entail sequential removal of the γ -glutamyl moiety and glycine from the glutathione conjugate, followed finally by N-acetylation of the resulting cysteine conjugate. It is important to recognize that GST enzymes are part of an integrated defense strategy, and their effectiveness depends on the combined actions of glutamate cysteine ligase and glutathione synthase to supply GSH and, on the other hand, the actions of transporters to remove glutathione conjugates from the cell (Hayes et al., 2005). Once formed, these conjugates are eliminated from the cell by the transmembrane MRP (multidrug resistance-associated protein).

1.22 Silymarin

Silymarin a known standardized extract purified from seeds of milk thistle i.e. Silybum marianum (Family: Composite) is composed of a mixture of isomeric flavonolignans: silibinin (its main, active component), isosilybin, silydianin, silychristin, Taxifolin and Quercetin (Crocenzi and Roma, 2006; El-Samaligy et al., 2006).

Silybin is the most biologically active component with regard to antioxidant and hepatoprotective properties.It is concentrated in the bile, achieving concentrations 60 times higher than that found in the serum.Silybin stimulates DNA polymerase, increasing the synthesis of ribosomal RNA and stimulating liver cell regeneration. It also stabilizes cellular membrane and increases the glutathione content of the liver (Kren and Walterova, 2005). Silybin acts as a free radical scavenger, increasing the activity of both superoxide dismutase and glutathione peroxidase in human cell lines (Kren and Walterova, 2005). It also inhibits the 5-lipoxygenase pathway in Kupffer cells, minimizing inflammation in the liver.

Silymarin is frequently used in the treatment of liver diseases where it is capable of protecting liver cells directly by stabilizing the membrane permeability through inhibiting lipid peroxidation (Mansour et al., 2006) and preventing liver glutathione depletion (Pradeep et al., 2007). These properties seem to be due to their ability to scavenge free radicals, to chelate metal ions and enhancing DNA polymerase(Borsari et al., 2001). It also prevents damage to the liver by antioxidative, anti-inflammatory, membrane stabilizing, immunomodulatory and liver regenerating mechanism. Seeds of S. marianum have been used for more than 2000 years to treat liver and gallbladder disorders, including hepatitis, cirrhosis and jaundice and to protect the liver against poisoning from chemicals, environmental toxins, snake bites, insect stings, mushroom poisoning and alcohol (Kren and Walterova, 2005). It is also reported to offer protection against chemical hepatotoxins such as CCl4, acetaminophen, phalloidin, galactosamine and thioacetamide and alcoholic liver diseases (Fraschini et al., 2002; Pradeep et al., 2007). Due to its proven hepatoprotective and antioxidant properties, silymarin is being used as a standard agent for comparison in the evaluation of hepatoprotective effects of plant principles (Dhiman and Chawla, 2005). Silymarin also has anticarcinogenic effects (Kang et al., 2004). Silymarin possesses a hydroxyl group at C5 in addition to the carbonyl group at C4, which may form a chelate with ferrous iron. This chelation can raise the activity to the level of most active scavengers, possibly by site specific scavenging (Abu Ghadeer et al., 2001). The free hydroxyl groups at C5 and C7 on the silymarin structure may also favour the inhibition of lipid peroxidation by reacting with peroxy radicals. This ability of silymarin leads to a significant increase in the cellular antioxidant defense machinery by ameliorating the deleterious effects of free radical reaction and increasing the content of GSH, which is important in maintaining the ferrous state (Abu Ghadeer et al., 2001; Ramadan et al., 2002).

1.23 Bilirubin

Bilirubin a reddish-yellow pigment carried in the plasma loosely bound to albumin is formed mainly from the breakdown of of haemoglobin in worn-out erythrocytes (Sharma and Biyani, 2012). It is also used by the liver to produce bile. The bound form of bilirubin which is not water soluble is usually referred to as INDIRECT reacting, free, prehepatic, or UNCONJUGATED bilirubin (McDonagh, 2007). On reaching the liver, the hepatocyte conjugates the indirect bilirubin with glucuronic acid and it is then referred to as DIRECT or CONJUGATED bilirubin which is water soluble. Direct bilirubin is excreted into the intestine via the biliary system. Some of the direct bilirubin is reabsorbed back into the circulation from the intestine. The direct bilirubin is not bound to albumin and is freely filtered by the glomeruli. The renal tubular epithelial cells readily reabsorb the filtered bilirubin in most animals.

When liver cells are damaged impairing its function or when biliary drainage is blocked, they may not be able to excrete bilirubin in the normal way, causing a build-up of bilirubin in the blood and extracellular (outside the cells) fluid. Some of the conjugated bilirubin also leaks out of the hepatocytes and appears in the urine, turning it dark amber. The presence of urine bilirubin indicates hepatobiliary disease (Singh et al., 2012). Serum bilirubin could be elevated if the serum albumin increases and the bilirubin shifts from tissue sites to circulation. Increased levels of bilirubin may also result due to decreased hepatic clearance and lead to jaundice and other hepatotoxicity symptoms (Saukkonen et al., 2006). Increase in bilirubin with little or no increase in ALT indicates cholestasis.

1.24 Serum/ Plasma Proteins

Plasma proteins represent a heterogeneous group with albumin constituting the major portion. Albumin which serves as a regulator of osmotic equilibrium is the major protein that the liver synthesizes and secretes into the blood (Mahajan, 2000). Low albumin levels indicate poor liver function. Albumin levels are usually normal in chronic liver diseases until cirrhosis and significant liver damage occur. Animal plasma normally contains 25-35 gm/L of albumin which constitutes 40 -60% of the total protein concentration (Gazuwa et al., 2012). In addition to albumin, plasma contains globulins, fibrinogen (removed from serum by the clotting process), glycoproteins, lipoproteins, acute phase proteins and transport proteins (Ewuola et al., 2014). Glycoproteinsand lipoproteins are the other major plasma proteins. Both of these serve as carriers of the substances bound to them.

Almost all proteins in the serum are produced by the liver. Immunoglobulins are the notable exception and they are produced by lymphoid tissue. Serum proteins are relatively short-lived with most having half-lives of about 10 days (Ewuola et al., 2014). The breakdown of these proteins occurs mostly in the liver with some catabolic activity in the intestine and kidney. Plasma and serum proteins, act as anions in acid-base balance, take part in coagulation reactions, and serve as carriers for many compounds.

Globulins are important serum proteins and they are primarily associated with antibodies.The globulin component is subdivided into important subfractions identified by electrophoresis as α-, β- and γ-globulins (Sharma et al., 2014). The α and β fractions are important carriers of lipids, lipid soluble hormones and vitamins. γ-globulins are primarily associated with antibodies. Conditions causing inflammation usually cause a measurable increase in serum levels of γ-globulins and often α-2 globulins (e.g. αALT). Fibrinogen is a plasma acute phase protein which is utilised in the coagulation process.

1.25 Phosphate

Phosphorus is an important ion most physiologically active as phosphate radical. Phosphorus is located in every cell of the body and is vitally concerned with many metabolic processes, including those involving the buffers in body fluids (Soetan et al., 2010). It is used in the structural proteins of cell wall, bone and other tissues and in active metabolic enzymes and pathways. Serum concentrations of phosphorus are regulated primarily by the renal tubules responding to parathyroid hormone stimulation (Moe, 2008). Calcium, magnesium and phosphorus levels are kept in a seesaw balance by the parathyroid hormones. A high phosphorus intake without a high calcium or magnesium intake causes calcium to leach from the bones and then leave the body with the urine. Increased tubular reabsorption occurs when the circulating parathyroid hormone level is decreased. Vitamin D enhances phosphorus absorption from the intestine and reabsorption from bone while serum levels are regulated by kidney reabsorption. The renal system is closely involved in the control of phosphorus levels and thus urea and creatinine are important adjunct determinations (Alfrey, 2004). Practically, every form of energy exchange inside living cells involve the forming or breaking of high-energy bonds that link oxides of phosphorus to carbon or to carbon-nitrogen compounds (Soetan et al., 2010; Murray et al., 2008).

1.26 Calcium

Calcium is one of the most important ions in the body utilised in bone and structural organisation, enzyme function, blood coagulation, in osmotic pressure and maintenance of fluid balances, and is also essential in muscle activity (Soetan et al., 2010). The majority of calcium in circulation exists as protein-bound and ionised calcium. Calcium in both forms is normally measured and reported as a total calcium value. When evaluating calcium, it is important to relate total calcium to the quantity of albumin in the serum and the acid-base status of the animal. The total calcium concentration can increase in hypoalbuminemia and decrease in hypoalbuminaemia. Acid-base changes alter the ratio of ionised to protein-bound calcium (Chase et al., 2000). Acidosis increases the ionised calcium fraction, whereas alkalosis increases the protein-bound fraction. Therefore total calcium, albumin and bicarbonate levels are important in evaluating calcium concentrations and related diseases.

Chemically induced hepatotoxicity may lead to the disruption of calcium homeostasis by increasing permeability of the plasma membrane, mitochondrial membrane and membranes of smooth endoplasmic reticulum thereby leading to increase in intracellular calcium (Singh et al., 2011). NADPH, a cofactor required by calcium pump may also decrease and this also leads to disruption of calcium homeostasis. Disruption of calcium homeostasis may result in the activation of many membrane damaging enzymes like ATPases, phospholipases, proteases and endonucleases, disruption of mitochondrial metabolism and ATP synthesis and damage of microfilaments used to support cell structure.

1.27 Sodium

Sodium is the primary ion in extracellular fluid. It, regulates plasma volume and acid-base balance; is involved in the maintenance of osmotic pressure of the body fluids; preserves normal irritability of muscles and cell permeability; activates nerve and muscle function and is involved in Na+/K+-ATPase; maintains membrane potentials; transmits nerve impulses and the absorptive processes of monosaccharides, amino acids, pyrimidines, and bile salts (Soetan et al., 2010). Its metabolism is regulated by aldosterone.A decrease or increase in the serum sodium level will have an effect on the plasma osmolality and this can have deleterious effects on the whole body – in particular, the central nervous system (Duggal et al., 2006). Any change in the serum sodium concentration not only changes the tonicity of the extracellular fluid, but also causes water to shift into or out of cells as the tonicity of the two compartments equilibrates (Duggal et al., 2006). This shift has important implications because the CNS manifestations of hypo- and hypernatraemia are the result of these water fluxes.

1.28 Potassium (K)

Potassium, the principal electrolyte (cation) present in intracellular fluid, functions in acid-base balance, regulation of osmotic pressure, conduction of nerve impulse, muscle contraction particularly the cardiac muscle, cell membrane function and Na+/K+-ATPase. It helps in the transfer of phosphate from ATP to pyruvic acid, is required during glycogenesis, it inhibits free radical formation and is critically important to maintaining normal heart rhythm and blood pressure (Soetan et al., 2010). There are reports that potassium levels in the body are critical to the stability of potassium ion channels, which are vital to the health of blood vessels. Sufficient potassium intake may help reduce vascular complications from diabetes, if only by improving high blood pressure.Aldosterone increases the secretion/excretion of both K and Mg (Huang and Kuo, 2007). The beneficial effect of a high-fiber diet on glucose metabolism may be due in part to the relatively large amount of potassium present in fruits, vegetables, and other high-fiber foods.

1.29 Magnesium

Magnesium, the second most abundant intracellular and the fourth most abundant cation in the body is the most important mineral for maintaining proper electrical balance and facilitating smooth metabolism in cells (Soetan et al., 2010). Magnesium is an active component of several enzyme systems in which thymine pyrophosphate is a cofactor. Oxidative phosphorylation is greatly reduced in the absence of magnesium. Magnesium is also an essential activator for the phosphate-transferring enzymes myokinase, diphosphopyridinenucleotide kinase, and creatine kinase. It also activates pyruvic acid carboxylase, pyruvic acid oxidase, and the condensing enzyme for the reactions in the citric acid cycle. It is also a constituent of bones, teeth, enzyme cofactor, (kinases, etc) (Soetan et al., 2000). It has a very good preventive effect against cancer and cured precancerous conditions such as leukoplasia, hyperkeratosis and chronic mastitis; essential for many enzyme reactions, especially in regard to cellular energy production, for the health of the brain and nervous system and also for healthy teeth and bones; it is an impressive infection fighter in the form of magnesium chloride; is used in protein synthesis, enzyme activation, oxidative phosphorylation, renal potassium and hydrogen exchange (Soetan et al., 2010). Mg primarily by attaching to phospholipids in membranes cell and its organelles reduce their permeability and enhance their function (Desai and Miller, 2012). It is required also as a cofactor in the various membrane ATP (energy requiring) pumps such as the Na/K pump, Ca/Mg, K/H and Na/H pumps; channels (such as Ca and Na) and exchangers (such as Na-Mg, Na-Ca and Na-H).

1.30 Zinc (ZN)

Zinc, an essential trace mineral in human nutrition is associated with more than 300 enzymatic systems. The role of zinc can be grouped into three general functional classes, namely catalytic, structural and regulatory. It is required for normal immune function; has antioxidant action; is a cofactor of many enzymes, including lactate dehydrogenase, alkaline phosphatase, and carbonic anhydrase; is a component of nuclear receptors for steroid, thyroid, and calcitriol hormones; is required for normal blood vessel response to changes in blood flow; is a membrane and cytoskeletal stabilizer; is an anti-apoptotic genes; is an important co-factor in DNA synthesis; is an anti-inflammatory agents; is used in the synthesis of insulin by pancreatic beta cells and in the action of insulin at the cellular level (Schultheiss et al., 2002). Free ions released from zinc during digestion, binds to endogenously secreted ligands before their transport into the enterocytes in the duodenum and jejunum (Tubek, 2007). The portal system then carries the absorbed zinc directly to the liver, and then released it into the systemic circulation for delivery to other tissues. About 70% of the zinc in circulation is bound to albumin, and any condition that alters serum albumin concentration can have a secondary effect on serum zinc levels.

1.31 Selenium (SE)

Selenium, an essential micronutrient for animals and humans plays an important role in antioxidant defense systems (Soudani et al., 2011). It plays a key role in the redox regulation and antioxidant function through glutathione peroxidases that remove excess of potentially damaging radicals produced during oxidative stress. It is present in the active center of glutathione peroxidase (GPx), which protects lipid membranes and macromolecules from oxidative damage produced by peroxides and also permits the regeneration of a membrane lipid molecule through reacylation (Heikal et al., 2012). Se also has the ability to counteract free radicals and protect the structure and function of proteins, DNA and chromosomes against oxidation injury.

Selenium is essential for optimum immune response and influences the innate and acquired immune systems by protecting the host from oxidative stress generated by the microbicidal effects of macrophages and during inflammatory reactions. The selenoenzyme thioredoxin reductase affects the redox regulation of several key enzymes, transcription factors and receptors, including ribonucleotide reductase, glucocorticoid receptors, anti-inflammatory protein AP-1, and nuclear factor-kappa B (NFkB), which binds to DNA and activates expression of genes encoding proteins involved in immune response (cytokines, adhesion molecules). Selenium mimics the action of insulin by activating key proteins that are also activated by insulin (Heikal et al., 2012).

1.32 Iron (Fe)

Iron, the most important transition element of the body, is found in functional forms in haemoglobin, myoglobin, the cytochromes, enzymes with iron sulphur complexes, and other iron-dependent enzymes (Sarkar et al., 2012). Iron exists in the blood mainly as haemoglobin in the erythrocytes and as transferrin in the plasma. It is transported as transferrin; stored as ferritin or haemosiderin and it is lost in sloughed cells and by bleeding (Soetan et al., 2010). In cellular respiration, it functions as essential component of enzymes involved in biological oxidation such as succinate dehydrogenase, catalase, peroxidase, cytochromes C, C1, A1, (Soetan et al., 2010). Iron is required for proper myelination of spinal cord and white matter of cerebellar folds in brain and is a cofactor for a number of enzymes involved in neurotransmitter synthesis. 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 function (Beard, 2001). Fe is required for making Hb and it is a prooxidant which is also needed by microorganisms for proliferation (Galan et al., 2005).

The plasma iron content is determined by the extent of blood losses, role of erythropoeisis, rate of apoferritin synthesis, rate of iron absorption from intestines and rate of red blood cell destruction. Deficiency disease or symptoms include anaemia, (hypochromic, microcytic). Early iron deficiency has also been reported to affect GABA metabolism in adult rats (Soetan et al., 2010). Brain is quite sensitive to dietary iron depletion and uses a host of mechanisms to regulate iron flux homostatically (Batra and Seth, 2002).

1.33 Breast Milk Toxicity

The content of breast milk has evolved over millions of years not only to provide nutrition but also to protect the offspring from infections and to induce immunological tolerance against common non-dangerous compounds. It is generally thought that each individual mother provides for the specific developmental needs of her individual child, which are rapidly evolving during the first months of life (M’Rabet et al., 2008).

Most compounds are fat soluble and tends to concentrate more in body fat. The levels of these compounds can be measured in many body tissues and fluids (blood, serum, urine, sperm, placenta, umbilical cord blood), but are easier to measure in breastmilk because of its higher fat content and the relative ease with which it can be extracted and analysed (Cattaneo, 2013). For this reason, breastmilk is generally used to measure the “body burden” of chemicals in human beings and we are often led to believe that it is the breastmilk itself, rather than the whole of our bodies, that is polluted with dangerous man-made chemicals. Prenatal exposure, especially if it occurs when the stem cells differentiate into cells developing into specific tissues and organs, may bring about harmful changes that can cause diseases later in life (Cattaneo, 2013).Because the brain starts developing during foetal life, and continues to grow and to develop rapidly after birth and during the first years of life, brain damage due to chemical residues can occur both in the pre- and the postnatal period.

After birth, the transfer of contaminants to the offspring continues through the rich,fatty breast milk. While nursing, a mammalian mother (including humans) draws down her fat stores, dumping not only the fat but also the persistent toxic chemicals she has accumulated in her body fat over the years into her milk. In this way, a load of contaminants that it has taken the mother decades to accumulate is passed onto her baby in a very short time (Cattaneo, 2013).

The composition of breastmilk is always changing depending on: the feeding session; how old the baby is; and on other factors like diet. During each feeding session, the foremilk which is the left over from previous feeding comes out first. The foremilk is quite watery because most of the fat in it has stuck to the cell membrane. The hindmilk which is creamier because it has full complement of fat and had no time to stick to cell membrane then comes later (MRabet et al., 2008). Breastmilk also changes from month to month, to meet up with the baby’s need. Colostrums made during late pregnancy and secreted during the first week after birth has more protein and antibodies; and less sugar and fat when compared to mature milk.

Colostrum and mature milk IgA and IgM are found in the form of IgA, or sIgA, and IgM much of which are produced by plasma cells in the mammary tissue. The plasma cells are part of the gut-associated lymphoid tissue (GALT). Lymphocytes from the GALT system migrate to the mammary gland providing antibodies specific for pathogens that may be encountered by the neonate (Hurley and Theil, 2011). Immunoglobulins are transported through the mammary epithelial cells by receptor-mediated mechanisms and transferred out of the mammary gland by milk ejection during suckling.Igs bind receptors at the basolateral surfaces of the mammary epithelial cell. These receptors are specific for the Fc portion of the immunoglobulin molecule. The receptor bound immunoglobulin in internalized via an endocytic mechanism, transported to the apical end of the cell and released into the alveolar lumen (Hurley and Theil, 2011).The immunoglobulin enters the GUT where it provides protective benefit for the neonate. IgG transfer, to the offspring in humans occurs during late pregnancy and provides the initial systemic source of that immunoglobulin. Infants consuming breast milk will primarily be consuming secretory IgA, which has significant protective activity in the intestine.To prevent excessive, destructive, and adverse immunological reactions between mother and foetus that might lead to ‘‘immune abortion,’’ the immune system of the foetus is actively down-regulated during pregnancy.

1.34 Disaccharides

Disaccharides namely sucrose, lactose and maltose are important sources of metabolic energy. The first step in their catabolism is their hydrolysis to their constituent monosaccharides. The enzymes responsible for the hydrolysis of sucrose, lactose and maltose are sucrase, lactase and maltase, respectively (Dilworth et al., 2005). These enzymes are located in the epithelial cells of the small intestine. The end-products resulting from the activities of these enzymes are actively translocated from the intestine to the blood by ATPases (Dilworth et al., 2005).

Lactose is a disaccharide found in mammalian’s milk. Cow’s milk and its various products form a part of man’s main daily food. Therefore, lactose forms a main part of one’s daily intake of carbohydrates. Beta-galactosidase hydrolyzes lactose into glucose and galactose, so it is commercially referred to as lactase, (Jokar and Karbassi, 2009). The main industrial application of beta-galactosidase is converting lactose to glucose and galactose. The following advantages are embodied in lactose hydrolysis: rapid fermentation of glucose; higher degree of sweetness of the liquid in which lactose has been hydrolyzed; higher solubility of glucose and galactose; higher stability of frozen condensed milk, in which lactose has been hydrolyzed; application of lactose hydrolyzed milk in cheese making results in rapid fall of pH and as a consequence rapid development of cheese flavor and texture takes place (Jokar and Karbassi, 2009), use of beta-galactosidase in whey eliminates technological problems (such as sandiness in whey powder and ice cream) improving the nutritional quality of whey and whey powder.

Some people cannot tolerate and digest lactose due to a lack of beta-galactosidase in their intestines. The consumption of milk and dairy products by these people leads to cramp, flatulence, vomiting, etc (Jokar and Karbassi, 2009). Therefore, one valuable source of nutrition would be unavailable for more than half of the people in the world due to lactose intolerance (Vasiljevic and Jelen, 2001). Since lactose intolerance is affecting a large proportion of the people (up to 50 million in USA), a cheap source of beta-galactosidase for the effective production of lactose hydrolyzed dairy products is of a substantial potential (Bury et al., 2001).

1.35 Botanical Outline of Senna mimosoides

Senna mimosoides formerly known as Cassia mimosoides belongs to the family Caesalpiniaceae and the genus Senna (Ekwueme et al., 2011a,b). It is known as sensitive senna and its leaves, pods and seeds are edible (Bruce, 2006). The genus Senna is a large and diverse group of annual and perennial herbs, shrubs, woody vines and trees found in the tropics and subtropics (Flory et al., 1992).Senna mimosoides is a weed common in wastelands, roadsides and fallows in savannah zone and is widespread in West Africa (Akobundu and Agyakwa, 1987). It is an erect, sometimes diffuse, annual or perennial shrub that reproduces from the seeds. It has a pithy stem which is woody at the base and is hairless or minutely hairy. The leaves are compound and alternate, with the petiole always bearing a gland below the bottom pair of leaflet. These leaflet are numerous, small, asymmetric and blunt at apex. The flowers are yellow and few. The fruit is a flat pubescent pod, about 6cm long and 5mm broad with 12-24 seeds. Senna species also include the following: S. alata, S. fistula, S. obtusifolia, S. occidentalis, S. roemeriana, S. folia, S. sophera, S. tora, S. obovata, S. angustifolia, S. auriculata, S. glauca, S. javanica, S. pumila, S.acutifolia (Joy et al., 1998).

Anthraquinones and related compounds have been known for years to occur in many Senna Species. It is well established that most of the anthraquinones in Senna are bound to sugars in the form of glycosides (with the aglycon occurring usually in a reduced form, e.g. anthrones, dianthrones, or anthrone) (Kuo et al., 2002). The major portion of the anthraquinones exists as the di- or tri-glycosides. These include rhein, physcion, obtusifolia, emodin, questin, lesser amounts of others, and chrysophanic acid. Chrysophanic acid made up virtually 50% of the total anthraquinones in the seed samples.Plants belonging to the family caesalpinia are known to posses the following constituents: a flavones glycoside 5, 3ˮ,4’ -tri-hydroxy-6-methoxy-7-o-alpha-L-rhamnopyranosyl-(1→2)-0-β-D-galactopyranoside (Yadav and Verma, 2003), 5-(2-hydroxyphenoxy methyl) furfural, (2’s)-7-hydroxy-5-hydroxymethyl-2-(2-hydroxypropyl)chromone, benzyl-2-hydroxy-3, 6-dimethoxy benzoate, benzyl-2-beta-o-D-glucopyranosyl-3,6-dimethoxybenzoate, 5-hydroxy-methylfurfural, (2’s)-7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (Kuo et al., 2002). They also contain dianthrones, anthranoids, hydroxyanthracene glycosides, mono-anthraquinone glycosides and aglika (Werner, 2007). Aglika are toxic substances but are not produced during cold maceration. According to Joy et al. (1998), leaves of senna are known to contain glucose, fructose, sucrose and pinitol. Mucilage consist of galactose, arabinose, rhamnose and galacturonic acid. Leaves also contain sennoside-c (8,8’-diglucoside of rhein-aloe-emodin-dianthrone). The pods contain sennoside A and B glycoside of anthraquinones rhein and chrysophanic acid. The seed contain β-sitosterol. Leaves and pods also contain 0.33% β-sterol and flavonols, kaempferol, kaempferol and isorhamnetin.

1.36 Previous Investigation Carried Out On Senna Species

Generally, the leaves of some senna are useful in treating constipation, abdominal disorder, leprosy, skin diseases, leucoderma, splenomegaly, hepatopathy, jaundice, helminthiasis, dyspepsia, cough, bronchitis, typhoid fever, anaemia, tumors (Joy et al., 1998).Traditionally, its roots, leaves, flowers and seeds are used as laxative and purgative. It is a vermifuge, anticonvulsant and used against chicken pox (Mann, 2003). Other uses include febrifuge, extrusion of guinea worm and black quarter (Ndi et al., 2000). Previous studies have shown that its leaves exhibited in vitro antibacterial, antimalarial and anti hepatotoxic properties. Seeds are brewed into a coffee like beverage for asthma and the flower infusion is used for bronchitis in the Peruvian Amazon (Akinloye et al., 2003). Senna mimosoides leaves are used in Nsukka folklore medicine, particularly Ukehe, Enugu State, Nigeria precisely to treat oedema in pregnant women and breastmilk toxicity in neonates. It is known to be a natural lipase inhibitor, i.e. it prevents the absorption of fats from digested food and is therefore used as a slimming agent (Ekwueme et al., 2011a,b). It also has a laxative effect i.e. it is used to relieve constipation and support normal body function (Ekwueme et al., 2011a,b). The aqueous leaf extract of Senna mimosoides exhibits anti-inflammatory effects by stabilizing membrane, inhibiting phospholipase A2 activity and prostaglandin synthase activity (Ekwueme et al., 2011a,b).

The bark of S. fistula has anti-inflammatory and antioxidant properties (Ilavarasan et al., 2005). It also possesses the following medicinal properties; it is cathartic, emetic, febrifuge, laxative and purgative. It is also used in the therapy of the following diseases, biliousness, bronchitis, fever, rheumatism and ringworm (Parekh and Chanda, 2007).S. occidentalis causes animal poisoning in different animal species. This poisoning specifically causes hepato myo encephalopathy (Vashishtha et al., 2007). It is also repulsive of morbid tumour (especially phlegm) and is a blood purifier, purgative, digestive and diaphoretic (Awan, 1984).

S. sophera has free radical scavenging activity. It is also repulsive of morbid tumour and is a blood purifier, purgative, digestive and diaphoretic (Awan, 1984). Ethnobotanical literature mentioned it in the treatment of pityriasis, psoriasis, asthma, acute bronchitis, cough, diabetes and convulsion (Kirtikar and Basu, 2000).

S.auriculata commonly known as Tanner’s cassia has its bark as astringent, the leaves and fruits are antihelminthic, seeds are used in eye troubles while the flower possesses anti-diabetic activity and is used as tea in diabetic patients and the root in skin disease (Siva and Krishnamurthy, 2005). It has also been used for the treatment of ulcer, leprosy and liver diseases (Kumar et al., 2002).

S. tora is traditionally used as laxative, purgatives, hepatoprotective, antidiabetic, antihelmintic, antioxidant, antimicrobial, bitter tonic and in wound healingliver disorders, skin and eye diseases (Rani and Satish, 2014).

1.37 Aim of the Study

This study is aimed at validating the traditional use of Senna mimosoides leaves in folk medicine for the treatment of oedema and breastmilk toxicity in neonates by investigating its immunological and biochemical nature.

1.38 Specific Research Objectives

The major objective of this study is to investigate the immunological and biochemical activity of the aqueous leaf extract. The research is designed to achieve the following specific objectives:

• To qualitatively and quantitativelydetermine the phytochemical compositionsof the leaves of S. mimosoides.

• To determine the acute toxicity (LD50) of the leaves of S. mimosoides in mice.

• To determine the effect of aqueous extract of S. mimosoides leaves on the immunomodulatory activities of the rats.

• To determine the effect of the aqueous extract of S. mimosoides leaves on cyclophosphamide-induced myelosuppresion.

• To determine the effect of the aqueous extract of S. mimosoides leaves on some haematological parameter.

• To determine the effect of the aqueous extract of S. mimosoides leaves on liver function marker enzymes on rats treated with CCl4.

• To determine the antioxidant properties of the aqueous extract of S. mimosoides leaves on rats treated with CCl4.

• To determine the effect of the aqueous extract of S. mimosoides leaves on lipid peroxidation in rats treated with CCl4.

• To determine the effect of the aqueous extract of S. mimosoides leaves on serum electrolyte concentrations in CCl4-induced hepatotoxicity in rats.

• To determine the damaging effect of CCl4 and ameliorative effect of the aqueous extract of S. mimosoides leaves on liver tissue using histopathological analysis.

• To determine the effect of the aqueous extract of S. mimosoides leaves on the activity of lactase.

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