ANTI-INFLAMMATORY AND HEPATOPROTECTIVE EFFECTS OF THE HOMOGENATE OF CUCUMIS SATIVUS (CUCUMBER) FRUITS

ABSTRACT

Research on  inflammation  has  become  the  focus  of global  scientific  study  because  of  its implication in virtually all human and animal diseases. Also, liver diseases have been on increase and of global concern.Cucumis sativus is believed to have anti-oxidant activity, high flavonoid content, anti-inflammatory and analgesic effect, which may be likely of use in the management of these diseases. The anti-inflammatory and hepatoprotective effects of the homogenate of Cucumis  sativus  fruit  were  therefore  studied.  The  fresh  fruit  of  Cucumis  sativus  was homogenized and used for all experimental analysis without further dilution. Acute toxicity tests of the homogenate of Cucumis sativusfruit were carried out. The phytochemical analyses and proximate compositions of the fruit homogenate were carried out. 1, 1-Diphenyl-2-Picryl Hydrazyl (DPPH) radical scavenging activity of the fruit  homogenate was determined. The effects of the fruit homogenate on agar-induced paw oedema in rats were investigated. The effects of the fruit homogenate on liver function enzyme (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) activities, total bilirubin concentration and  lipid profile (total cholesterol, high density lipoprotein, triacylglycerol and low density lipoprotein concentrations)  in  rats  intoxicated  with  carbon  tetrachloride  (CCl4)  were  evaluated  using standard biochemical methods.The effects  of the  fruit  homogenate on hypotonicity-induced haemolysis of RBC, phospholipase A2and prostaglandin synthase activitieswere also studied. Data were analysed using SPSS and two-way ANOVA; the acceptance level of significance was p˂0.05.The qualitative phytochemical tests on the homogenate of Cucumis sativus fruitrevealed the  presence  of  flavonoids,  alkaloids,  terpenoids,  glycosides,  resins,  steroids,  saponins and tannins. The quantitative phytochemical analysis of the homogenate ofCucumis sativus fruit showed that,reducing sugars (574.36 ± 3.88 mg/g) was highest amount when compared to other phytochemicals,  alkaloids  (2.22  ±  0.96  mg/g)  and  flavonoids  (2.14  ±  0.56  mg/g)  were moderately present while cyanogenic glycoside (0.21 ± 0.13 mg/g) was the lowest in quantity.Proximate analysis showed thatCucumis sativus fruit contained the following – fibre (1.30  ±  0.01%),  moisture  (94.6  ±  0.08%),  protein  (3.11  ±  0.07%)  and  ash  (1.07  ±

0.24%)contents. The acute toxicity test showed no toxicity up to 5ml/kg (≡ 5000mg/kg) body weight which indicated the possible safety of the fruit to the users. There was relative increase in the percentage inhibition of DPPH radical scavenging activity with increased amount of the homogenate. At doses of 2ml and 4ml/kg b.w., the fruit homogenate significantly (p ˂ 0.05) inhibitedagar-induced raw paw oedema relative to control. Studies on membrane stabilization using hypotonicity-induced red blood cell haemolysis revealed that the fruit homogenate significantly (p˂0.05)inhibited haemolysis when compared to indomethacin (a known standard drug).The homogenate exhibited a significant (p˂0.05) dose (0.5ml and 1.0ml) related inhibition of prostaglandin synthase activity (79.9% and 81.0% respectively), compared to 0.4mg/ml of indomethacin, standard drug  (82.0%). The  fruit  homogenate like  prednisolone significantly (p˂0.05) inhibited phospholipase A2 activity.Treatment of rats with the homogenate of Cucumis sativus fruits significantly (p˂0.05) decreased CCl4-inducedelevated levels of the liver enzymes ALT, AST and ALP and of total bilirubin in the serum when compared to positive control. The homogenate also attenuated the CCl4-induced elevation of LDL, total cholesterol and triacylglycerol amounts and ameliorated the induced depletion of HDL. The results indicated that the homogenate of Cucumis sativus fruits possesses anti-inflammatory activities and hepatoprotective effects.

CHAPTER ONE

INTRODUCTION

In most rural communities of developing countries, plant materials are sources of shelter, food and  medicinal  compounds  (Oduolaet  al.,  2005).  Herbal  medicine  is  fast  emerging  as  an alternative treatment to available synthetic drugs for the treatment of disease possibly due to lower cost, availability, fewer adverse effect and perceived effectiveness (Ubakaet al., 2010). The World Health Organization (WHO) has shown great interest in plant derived medicines which have been described in the folklore medicines of many countries (Mukherjee, 2002). However, the historic role of medicinal plants in the treatment and prevention of diseases and their  role  as  catalyst  in  the  development  of  pharmacology do  not  assure  their  safety  for uncontrolled use by an uninformed public (Matthew et al., 1999). It is thus, imperative that plant products, which have been used from ages, have scientific support for their efficacy. Medicinal plants with anti-inflammatory activity are considerably employed in the treatment of several inflammatory disorders (Iwuekeet al., 2006). Research on inflammation has become the focus of global scientific study because of its implication in virtually all human and animal diseases. Many anti-inflammatory plants and agents modify inflammatory responses by accelerating the destruction or antagonizing the action of the mediators of inflammatory reaction (Anosike et al.,

2009). The inflammatory responses involve a complex array of enzyme activation, mediator release,  fluid  extravasations,  cell  migration,  tissue  breakdown  and  repair.  These  different reactions in the inflammatory response cascade are therapeutic targets which anti-inflammatory agents including medicinal plants interfere with to suppress inflammatory responses usually invoked in such disorders as rheumatoid arthritis, osteoarthritis, in infection or injury (Abebe, 2002).1.1 Inflammation

The process of inflammation is one of the most essential reactions of cells and tissues to injury among the key homeostatic mechanisms of higher organisms. Sanderson (1971) defined inflammation as the succession of changes which occur in a living tissue when it is injured. On the other hand, inflammation can be defined as a response of the tissue to an infection, irritation or foreign substance (Guyton, 1981). It is a part of the host’s defence, but if the response becomes great, it may be worse than the disease state that elicited the response. In extreme cases, the response becomes fatal. As a defensive reaction, inflammation is useful to the body but, often leads to tissue damage. Though inflammation is a defence mechanism, the complex events and mediators involved in the inflammation reactions can induce, maintain or aggravate many diseases (Sosa et al., 2002). Inflammation is part of our innate immunity. Our innate immunity is what is naturally present in our bodies when we are born, and not the adaptive immunity we get after an infection or vaccination. Innate immunity is generally non-specific, while adaptive immunity is specific to one pathogen. Inflammation is a mechanism of innate immunity. The body immune system (defence system) triggers an inflammatory response in autoimmune diseases, when there are no foreign substances to fight off, causing damage to its own tissues (Coussens and Werb, 2002).  Inflammation gets rid of any irritant either by flushing out or diluting the irritant with the fluid produced. Inflammation also takes care of irritants produced by antibody of some of the infiltrating cells and finally by phagocytosis. It rids the body of foreign matter, disposes damaged cells and initiates wound healing.

1.2 Classification of inflammation

Inflammatory reaction can be classified based on the type of exudates produced by the body. These are serous type, in which the serous fluid produced is used to flush out the invading irritant; mucus type, in which watery slimy fluid produced is to get rid of the invading irritant and fibrinous type, where the exudates contain fibrin that cover the area being irritated. Also, haemorrhagic inflammation exists, when there is production of bloody fluid to fight the irritant. Others are purulent and proliferative types that  are characterized  by formation of pus and connective tissues respectively.

On the other hand, it could be classified as either acute or chronic, depending on the type and duration  of  the  antigen  challenge  and  is  mediated  by  some  chemical  substances  such  as histamine, serotonin, slow reacting substances of anaphylaxis (SRS-A), prostaglandins and some plasma enzyme systems such as the complement system, the clotting system, the fibronolytic system and kinin system.

1.2.1   Acute inflammation

Acute inflammation is usually of sudden onset, and characterized by the classical signs in which the vascular and exudative processes predominate (Dorland, 1982). It is the initial response of the body to harmful stimuli produced by the infiltration of plasma and leukocytes from the blood into an injured tissue. Histologically, acute inflammation is characterized by a complex series of events which include: vasodilation of the blood vessels leading to excess local blood flow, increased capillary permeability leading to leakage of fluids and blood into the interstitial space, clotting of the fluids in the interstitial space because of excess fibrinogen leaking from the capillaries, leukocyte migration (granulocytes and monocytes) into the injured area and swelling of the tissue cells (Ferrero-Milianiet al., 2007). As long as the injurious stimulus is present, acute inflammation occurs and ceases once the stimulus has been removed, broken down, or walled off by  scarring  (fibrosis).  Three  main  processes  occur  before  and  during  acute  inflammation; Dilation of arterioles,increased permeability of the capillariesand migration of neutrophils, and possibly some macrophages out of the capillaries and venules.When the skin is scratched (and is not broken), one may see a pale red line. Soon the scratched area goes red; this is because the arterioles have dilated and the capillaries have filled up with blood and become more permeable, allowing  fluid  and  blood  proteins  to  move  into  the  space  between  tissues.This  is  further explained in figure 1 below.

Figure 1: Overview of vascular changes in acute inflammation

Acute inflammation is characterized by five cardinal signs – “PRISH”, that is;

Pain –  The  inflamed area  is  likely to  be painful, especially when touched. Chemicals that stimulate nerve endings are released, making the area much more sensitive.

Redness – This is because the capillaries are filled up with more blood than usual. Immobility – There may be some loss of function.

Swelling – Caused by an accumulation of fluid.

Heat – more blood in the affected area makes it feel hot to the touch.

These signs develop as an acute response to  a local inflammatory insult  by the  action of inflammatory   mediators   (Garrison,   2000).   The   inflammatory   insults   may   be   caused mechanically, e.g. by the presence of foreign bodies, or chemically, e.g. by toxin, acidity and alkalinity,  or physically, e.g.  by temperature, or by internal processes, e.g. uraemia, or by microorganisms, e.g. bacteria, viruses and parasites. The pain is often attributed to increased pressure on the nerve endings, the irritating effects of toxic products and the action of certain mediators of the inflammatory process. The redness is caused by an increase of blood volume in the inflamed area; the swelling is due to increase of blood and additional presence of substances which exude from the blood vessels (exudates) into the surrounding tissues. The heat results from the increased flow of blood. These five acute inflammation signs are only relevant when the affected area is on or very close to the skin. When inflammation occurs deep inside the body, such as an internal organ, only some of the signs may be detectable. Some internal organs may not have sensory nerve endings nearby, so there may be no pain, as is the case with some types of pneumonia(acute inflammation of the lung). If the inflammation from pneumonia pushes against the parietal pleura (inner lining of the surface of the chest wall), then there is pain. The desirable outcome of acute  inflammation process,  which at  least  initially  is  protective and homeostatic, is the isolation and destruction of the injurious agent and resolution of the inflammatory lesion so that normal tissue conditions are fully restored. If however, the challenging stimulus persists, the inflammation may become chronic and the micro-circulating changes characteristic of acute inflammation is replaced by lesions typical of the chronic-disease (Serhan, 2008). This is further explained in figure 2 below.

Figure 2: Schematic representation of inflammatory action

1.2.2 Chronic inflammation

Chronic inflammation is of slow progress and is marked chiefly by the formation of connective tissues. It may be a continuation of an acute form or a prolonged low grade form and usually causes permanent tissue damage (Dorland, 1982). It is characterized by simultaneous active inflammation,  tissue  damage  and  attempts  at  healing  (repair)  of  the  tissues  from  the inflammatory process  (Eminget  al.,  2007).  Chronically  inflamed  tissue  is  characterized  by infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes and plasma cells) tissue destruction and attempts at healing which include angiogenesis and fibrosis. These mononuclear immune cells are powerful defensive agents in the body, but the toxins they release (including reactive oxygen species) are injurious to the organism’s own tissues as well as to the invading agents. Consequently, chronic inflammation is almost always accompanied by tissue damage (Insel, 1996).

1.3 Inflammatory responses

1.3.1 Acute vascular response

Acute vascular response is the earliest gross event of response to injury by a transient arteriolar vasoconstriction, the narrowing of the blood vessels, which is caused by contraction of the smooth muscles of the blood vessel walls. It is seen on the skin as a transient blanching, the whitening of the skin. Acute vascular response which is a characteristic of acute inflammation, also includes vasodilation, increased permeability and increased blood flow, all of which are induced  by the  actions of various inflammatory mediators. Vasodilation occurs first  at  the arteriolelevel, progressing to the capillary level, and brings about a net increase in the amount of blood present, causing the redness and heat of inflammation.

Increased permeability of the vessels results in the movement of plasma into the tissues, with resultant stasisdue to the increase in the concentration of the cells within blood – a condition characterized by enlarged vessels packed with cells. Stasis allows leukocytesto marginate (move) along the endothelium, a process critical to their recruitment into the tissues. The movement of plasma fluid, containing important proteinssuch as fibrin and immunoglobulins (antibodies), into

inflamed tissue, is achieved via the chemically induced dilation and increased permeability of blood vessels, which result in a net loss of blood plasma. The increased collection of fluid into the tissue causes it to swell (oedema). This extravasated fluid is funnelled by lymphaticsto the regional lymph nodes,  flushing  bacteria  along  to  start  the  recognition and  attack  phase of theadaptive immune system. Normal flowing blood prevents this, as the shearing forcealong the periphery of the vessels moves cells in the blood into the middle of the vessel. Acute vascular response follows within seconds of tissue injury and lasts for some minutes (Stvrtinovaet al.,

1995).

1.3.2 Acute cellular response

Acute cellular response occurs in inflammation if there has been sufficient damage to the tissues or if infection has occurred. It  takes place over the next  few  hours. During acute cellular response, inflammatory cells (mainly neutrophils) are normally contained in the central or axial part of the blood volume, thereby appearing in the tissues and attaching to the endothelial cells within the blood vessels, then crossing over into the surrounding tissue (diapedesis). The process is facilitated by the stasis of the blood. As a result of stasis, the red and white blood cells tend to come together. In this response, erythrocytes may leak into the tissues and haemorrhage can occur (e.g. a blood blister). If the blood vessel is damaged, fibrinogen and  fibronectin are deposited at the site of injury, platelets aggregate and become activated, and the red cells stack together in what is called “rouleau” to help stop bleeding and aid clot formation. The dead and dying cells contribute to pus formation (Stvrtinovaet al., 1995).

1.3.3 Chronic cellular response

When the tissue damage is severe and occurs over few days, chronic cellular response may follow.  In  it,  mononuclear cell  infiltrate,  composed of macrophages and  lymphocytes. The macrophages involved are activated by several cytokines, including IL-2 (interleukin-2), and their migration is stimulated by components of the extracellular matrix (i.e. collagen, elastin, fibronectin), TGF-β,  and  complement  cascade  products. They are  the  most  important  cells present in the later stages of the inflammatory process (48-72 hours), acting as the key regulatory cells for healing and repair. Their subsequent production of inflammatory cytokines (IL-1 and

TNF [tumour necrosis factor]) and growth factors (mainly TGF-β and PDGF) appears to be the most critical cell-driven event of the entire phase of inflammation. Releasing these products into the wound recruits fibroblasts, keratinocytes and endothelial cells to repair the damaged blood vessels. The absence of macrophages is associated with failure to progress to normal fibroblast recruitment and function.

Macrophages are  also  capable  of releasing  proteolytic enzymes (e.g.  collagenase) that  can debride  tissue  and  extracellular  matrix.  Apart  from  wound  debridement,  other  important functions of these cells include nitric oxide synthesis, phagocytosis of bacteria, and stimulation of angiogenesis (Doherty et al., 2002). Additional growth factors such as TGF-α, HB-EGF (heparin-binding EGF), and bFGF (basic fibroblast growth factor), secreted by both PMNs and macrophages, may further stimulate the inflammatory response. On the other hand, the depletion of circulating monocytes and tissue macrophages can cause severe changes in wound healing, leading to poor wound debridement, delayed fibroblast proliferation, inadequate angiogenesis and poor fibrosis (Enoch and Price, 2004).The last cell type to enter the wound during the inflammatory phase (>72 hours after injury) is the lymphocyte. Lymphocytes may be attracted by IL-1, IgG and complement products. Since IL-1 is believed to play a key role in the regulation of collagenase, lymphocytes may be involved in collagen and extracellular matrix remodelling (Sedgwick et al., 1981).

1.3.4   Resolution

Inflammation is often considered in terms of acute, inflammation involving all acute vascular and  acute cellular  response, and  chronic  inflammation,  involving all  the  events of chronic cellular response and resolution or scarring. Resolution, that  is restoration of normal tissue architecture may occur over the following few weeks after a tissue injury. It involves the removal of blood clots by fibrinolysis and if the tissue cannot be returned to its original form, it will result to scarring from in-filling with fibroblasts, collagen and new endothelial cells. Any infectious agent that was not completely destroyed and removed from the site of injury, would be walled off from the surrounding tissues in granulomatous tissue (Serhan and Savil, 2005). Resolution of inflammation occurs by different mechanisms in different tissues (Eminget al., 2007). Mechanisms which  serve to  terminate  inflammation include;short half-life  of  inflammatory

mediators in-vivo, production and release of transforming growth factor (TGF) beta from macrophages (Ashcroft, 1999) and production and release of interleukin 10 (IL-10) (Sato et al.,

1999).  Serhan, 2008  reported production of anti-inflammatory lipoxins  as  a  mechanism of termination of inflammationOther mechanisms include; down regulation of pro-inflammatory molecules, such  as  leukotrienes, up  regulation  of anti-inflammatory molecules such as  the interleukin  1  receptor  antagonist  or  the  soluble  tumour  necrosis  factor  receptor  (TNFR), apoptosis of pro-inflammatory cells, desensitization of receptors (Greenhalgh, 1998), production of resolvins, protectins or maresins, down regulation of receptor activity by high concentrations of ligands, increased survival of cells in regions of inflammation due to their interaction with the extracellular matrix (ECM) (Tender et al., 2002; Jiang et al., 2005). Cleavage of chemokines by matrix metalloproteinase (MMPs) might lead to production of anti-inflammatory factors (McQuibbanet al., 2000).

Acute inflammation normally resolves by mechanisms that have remained somewhat elusive. Emerging evidence now suggests that an active, coordinated programme of resolution initiates in the first few hours after an inflammatory response begins. After entering tissues, granulocytes promote the switch of arachidonic acid–derived prostaglandins and leukotrienes to lipoxins, which initiate the termination sequence. Neutrophil recruitment thus ceases and programmed cell death by apoptosis is engaged. These events coincide with the biosynthesis, from omega-3 polyunsaturated fatty acids, of resolvins and protectins, which critically shorten the period of neutrophil infiltration by initiating apoptosis. Consequently, apoptotic neutrophils undergo phagocytosis by macrophages, leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines as transforming growth factor-β1. The anti-inflammatory programme ends with the departure of macrophages through the lymphatics (Serhan and Savil, 2005).

1.4 Inflammatory cells

The different numbers of cells responsible for inactivation and removal of invading infectious agents and damaged tissues are recruited into area where there is a tissue damage, and they differ depending on the phase of the inflammation (which is mostly second and third phase), the type of inflamed tissue and factors triggering the inflammatory process (Anosike, 2010). In acute inflammation, neutrophils are the main cells involved. A pyrogenic bacterial infection occurs and

local  depositions  of  immune  complexes  containing  IgG  are  the  cause  of  inflammation, neutrophils are the dominant cells (Wagner and Rothz, 2000).   Fehervariet al.(2005) reported that the mononuclear phagocytes are the main infiltrating cells in sub-acute and chronic phase of inflammatory reactions and in cases of infection with intracellular parasitic microorganisms, and the eosinophils and basophils are predominant when inflammation is triggered by immediate allergic reactions or parasites. Lymphocytes are involved in specific immune responses and endothelial cells function in the regulation of leukocyte emigration from the blood into the inflamed tissue while platelets and mast cells are involved in the production of early phase mediators (Stvrtinovaet al., 1995).

1.5Oxidative damage in inflammation

During inflammation, free radicals are produced by neutrophils, phagocytes, macrophages, endothelial and other cells.  Upon activation, neutrophils and  mononuclear phagocytes have increased oxygen consumption, during which they release lysozyme and reactive oxygen species (ROS). These reactive oxygen species cause oxidative damage to biological tissues and are implicated in the development of inflammatory and other chronic disease conditions such as atherosclerosis, stroke and even ageing (Halliwellet al., 1992). ROS include both oxygen free radical (superoxide radical (O2), hydroxyl radical (OH), alkoxy radical (RO), peroxyl radical (ROO),  hydroperoxyl  radical  (HOO)  and  oxygen  non-radical  that  are  reactive  (hydrogen peroxide H2O2), hypochlorous acid (HOCl), ozone (O3) and singlet oxygen (O2) (Babu,et al.,

2002). Upon activation of the respiratory burst, oxygen is univalently reduced by NADPH

oxidase to superoxide anion, which is then catalytically converted by the action of superoxide dismutase to hydrogen peroxide. Hydrogen peroxide interacts with myeloperoxidase (MPO) contained in neutrophils azurophil granules to produce hypochlorous acid which is metabolised to  hypochlorate  and  chlorine.  Hydroxyl  radical  and  hypochlorate  are  the  most  powerful substances involved in microbiocidal and cytotoxic reactions.

1.6 Antioxidants

Antioxidants are complex and diverse group of molecules that protect key biological sites from free-radical induced oxidative damage. They are molecules capable of showing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that  damage cells.  Antioxidants terminate these  chain reactions by removing free radicals intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents. They can act by removing: oxygen or decreasing local oxygen concentration, catalytic  metal ions,  key ROS such as  superoxide radical  and H2O2and by breaking the chain of initiated free radical sequence (Sies, 1997). Antioxidants are classified into two broad divisions: Water soluble antioxidants which react with oxidants in the cell cytoplasm and lipid soluble antioxidants which protect cell membranes from lipid peroxidation (Sies, 1997). Antioxidants have anti-inflammatory properties. By scavenging radicals, they reduce inflammatory signals, thus reducing inflammation.

1.7 Inflammatory disorders

Inflammatory abnormalities are a large group of disorders which underlie a vast variety of human diseases. The immune system is often involved in inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation. Non-immune diseases with aetiological origins in inflammatory processes include cancer, atherosclerosis, and ischaemic heart disease (Contranet al., 1999).A large variety of proteins are involved in inflammation, and any one of them is open to a genetic mutation  which  impairs  the  normal  function  and  expression  of  that  protein.  Examples  of disorders associated with inflammation include: acne vulgaris, asthma, autoimmune diseases, coeliac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis and interstitial cystitis.

1.8 Anti-inflammatory agents

Anti-inflammatory agents are compounds which act by several mechanisms to inhibit the various changes leading to  inflammation. They  modify inflammatory responses by accelerating the destruction or antagonizing the action of the mediators of the inflammatory reaction (Anosike et al., 2009). They are divided into steroidal and non-steroidal anti-inflammatory agents. Steroidal

anti-inflammatory drugs are the glucocorticoids, they bind to cortisol receptors; they are called corticosteroids. Glucocorticoids suppress the expression of cyclooxygenase (COX-2), and thus prostaglandin production. This contributes to its anti-inflammatory effect. Examples of glucocorticoids includecortisone, cortisol, corticosterone, hydrocortisone, triamcinolone,betamethasone prednisone and prednisolone. Non-steroidal anti-inflammatory drugs which include aspirin, ibuprofen, diclofenac, and indomethacin are used primarily for the treatment of inflammatory diseases such as rheumatoid arthritis, pain and fever. They may act via single or combination of any of the mechanisms involving; inhibition of arachidonic acid metabolism,   inhibition   of   cyclooxygenase   (COX)/Inhibition  of   prostaglandin   synthesis, inhibition of lipoxygenase (LOX), inhibition of cytokines (IL, TNF), inhibition of leukocyte migration/phagocytosis, uncoupling  oxidative  phosphorylation,  release  of steroidal  hormone from the adrenals and stabilization of lysosomal membrane (Wallace, 2002)

1.8.1 Stabilization of lysosomal membrane

During inflammation, the lysosomes lyse to release their component enzymes which produce a variety of disorders. Since human red blood cell membranes are similar to lysosomal membranes (Goodman et al., 1982; Gandhisanet al., 1991), human red blood cell membrane stabilization has, therefore been used as a method to study the mechanism of action of anti-inflammatory drugs (Seeman, 1968; Murugeshet al., 1981). Stabilization of lysosomal membranes is important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated neutrophil such as bactericidal enzymes and proteases, which cause  further tissue inflammation and damage upon extracellular release (Chou, 1997). Some NSAIDs like indomethacin and acetylsalicylic acid are known to possess membrane stabilization properties (Murugeshet al., 1981; Furst and Munster, 2001) which may contribute to the potency of their anti-inflammatory effect.

Hypotonicity-induced haemolysis of red blood cells occurs due to osmotically coupled water uptake by the cells, and leads to swelling and lysis, resulting in the release of haemoglobin, hencehaemolysis.  Haemolysis  is  a  reflection  of  the  stability  of  red  blood  cell  membrane (Iwuekeet  al.,  2006).The  vitality  of  cells  depends  on  the  integrity  of  their  membranes (Weissman, 1967).Feirraliet al. (1992)reported that the exposure of red blood cell to injurious

substances such as hypotonic medium, heat, methyl salicylate and phenyl hydrazine results in lysis  of  its  membrane  accompanied  by  haemolysis  and  oxidation  of  haemoglobin.  The haemolytic effect of hypotonic solution is related to excessive accumulation of fluid within the cell resulting in the rupturing of its membrane. Such injury to RBC membrane will further render the cell more susceptible to secondary damage through free radical-induced lipid peroxidation. This notion is consistent with the observation that the breakdown of biomolecules lead to the formation of free radicals which in turn enhance cellular damage (Maxwell, 1995; Halliwell and Whiteman, 2004). The progression of bone destruction seen in rheumatoid patient for example, has been shown to be due to increased free radical activity (Pattison et al., 2004). It is therefore expected that compounds with membrane-stabilizing properties, should offer significant protection of cell membrane against injurious substances (Perenzet al., 1995; Shindeet al., 1999).

Compounds with membrane-stabilizing properties are well known for their interference with the early phase of inflammation reactions, namely the prevention of the release of Phospholipases that trigger the formation of inflammatory mediators (Aitadafounet al., 1996). The stabilization of the red blood cell membrane prevents the release of lytic enzymes and active mediators of inflammation, such as 5-hydroxytrptamine, histamine and kinins (Phillips and Morrison, 1970).

1.8.2 Phospholipase A2

The major structural feature of cell membrane is the lipid bilayer. The lysosomal enzyme, phospholipase A2hydrolyses saturated or unsaturated lecithin dispersed as liposomes (Lewis et al., 1979). Phospholipase A2 is an enzyme that releases fatty acids from the second carbon group of glycerol. This particular phospholipase specifically recognizes the Sn-2 acyl bond of phospholipids and catalytically hydrolyses the bond releasing arachidonic acid and lysophospholipids. Lysophospholipids are  powerful detergents that  disrupt  cell  membranes, thereby lysing cells. Upon downstream modification by cyclooxygenases (Cox) – (an enzyme that is responsible for the formation of prostanoids. The three main groups of prostanoids – prostaglandins,prostacyclins, and thromboxanes are  involved in the  inflammatory response). Arachidonic acid is modified into active compounds called eicosanoids. Eicosanoids include prostaglandins and leukotrines, which are categorized as inflammatory mediators (Dennis, 1994). In other words, the major action of the phospholipase A2, an acyl hydrolase, during inflammation is to cleave from membrane phospholipids free fatty acids some of which are necessary precursors of prostaglandins. The activity renders the membrane leaky and the contents of the red blood cells flow out.

1.8.3 Prostaglandin synthase/Cyclooxygenase

Prostaglandins are important lipid mediators derived from arachidonic acid that control not only numerous physiological events such as blood pressure, blood clotting and sleep but also inflammation (Funk, 2001). Prostaglandin E2 is a key player in pyresis, pain and inflammatory responses and the beneficial therapeutic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are essentially attributed to the suppression of prostaglandins E2 (Funk, 2001). The biosynthetic  pathway  to  prostaglandin  E2   includes  the  release  of  arachidonic  acid  from membrane phospholipids by phospholipases A2  followed by conversion via Cox-1 and -2 to prostaglandins H2  and  its  subsequent  isomerization by prostaglandins E2  synthases (PGEs). mPGEs1is induced by pro-inflammatory stimuli such as interleukin-1β (IL-1β) or lipopolysaccharide (LPS), and receives PGH2 preferentially from Cox-2 (Murakami et al., 2002). Thus inflammation, pain, fever and different types of cancer are closely linked to the increased prostaglandin E2 formation originating from up-regulated MPGEs1 (Samuelsson et al., 2007).

Prostaglandin E2 is produced in small quantity under normal physiological conditions but in large quantity during inflammation (Girloyet al., 1999). The substantial increase in prostaglandins E2 in inflammation is attributable to expression of Cox-2. Its (Prostaglandin E2) implication in the majority of inflammatory reactions including pain, increased capillary permeability, vascular dilation and recruitment of inflammatory cells. Their ability to increase vascular permeability in man and animals and their ability to cause leucocyte emigration (Crook et al., 1976), suggest that they are important mediators of the acute phase of the inflammatory reaction. The reversal of the inflammatory reactions is caused by the inhibition of the biosynthesis by anti-inflammatory agents, the ability to inhibit the biosynthesis of prostaglandins E2 underlie suppression of pain (analgesic), reversal of vasodilation, decreased capillary permeability and inhibition of migration of inflammatory cells. (Aba and Mensah-Attipoe, 2008). In addition to their involvement in the inflammatory response, prostaglandins sensitize the skin to painful stimuli probably because they sensitize pain receptors to mechanical and chemical stimulations (Roberts and Morrow, 2001) such as the pain-producing effect of mediators (e.g. histamine, kinins etc.) which are released in tissue injury and inflammation.

Prostaglandins manifested during strong physiological effects include regulating the contraction and relaxation of smooth muscle tissue (Nelson and Randy, 2005). Produced by almost all nucleated cells, prostaglandins are autocrine and paracrine lipid mediators that act upon platelets, endothelium, uterine and mast cells, synthesized in the cell from essential fatty acids.Prostaglandins level are increased by cox-2 in scenario of inflammation.

The production of prostaglandins begins with the formation of a cyclopentane ring in the linear fatty acid, as catalysed by prostaglandin H2 synthase. The heme-containing enzyme (Commonly called COX, not to be confused with cytochrome c oxidase, which is also called COX) contains two catalytic activities: a cyclooxygenase that adds two molecules of O2 to arachidonate, and a peroxidase that converts the resulting hydroperoxy group to an OH group.

Some of the major uses of prostaglandins include; induction of labour, regulation of calcium movement,  control  of  hormone  regulation,  control  of  cell  growth,  decreasing  intraocular pressure. Also, they act on thermoregularity centre of the hypothalamus to produce fever and on mesangial cells in the glomerulus of the kidney to increase glomerular filtration rate and on the pariental cells in the stomach wall to inhibit acid secretion, so as to maintain the integrity of the gastric lining of the stomach. They also sensitize spinal neurons to pains.

Inhibition of biosynthesis of prostaglandins and enzyme – cyclooxygenase

Cyclooxygenase (COX) is the pivotal enzyme in prostaglandin biosynthesis. It exists in two isoforms; constitutive COX-1 which is responsible for physiological functions and makes prostaglandins that protect the stomach and kidney from damage and inducible COX-2 which is involved in inflammation, inducing inflammatory stimuli such as cytokines and produces prostaglandins that contribute to the pain and swelling of inflammation. COX-2 is thought to be involved in ovulation and labour.

Inhibition of COX explains both the therapeutic effects (Inhibition of COX-2) and side effects

(Inhibition of COX-1 of Non-Steroidal Anti-Inflammatory Drugs, NSAIDs) (Vane and Botting,

1996). It  is  important to  note, that  non-steroidal anti-inflammatory drugs which selectively inhibit  COX-2  arelikely  to  retain  maximal  anti-inflammatory efficacy  combined  with  less toxicity. The two isoforms (COX-1 and COX-2) share a high degree (60%) of sequence identity and structural homology (Voet et al., 2013).

1.9Anti-inflammatory plants

The practice of traditional medicine is as old as the origin of man.   The use of plants in traditional medicine referred to as herbalism or simply botanical medicine (Edeogaet al., 2005) falls outside the mainstream of the Western or orthodox medicine. In the field of ethnomedicinal plants or plants used as anti-inflammatory agents, a lot of information is available. Bagulet al.(2005)  reported  the  anti-inflammatory activity  of  two  Ayurvedic  formulations. Bahattacharyaet al.(2005) reported the anti-inflammatory potential of methanol extract of Stepeniaglabraof  Menispermaceae  family.  Ammaret  al.(1997)  reported  anti-inflammatory activity  of  bioactive  fractions  isolated  from  seeds  of  TrigonellafoenumgraciumL., roots  of GlycyrhizaglabraL. and fruits of CoriandrumsativumL. The anti-inflammatory and anti- ulcerogenic activity of ethanol extract of Zingiberofficinalewas demonstrated by Anosike et al. (2009). Iwuekeet al. (2006) demonstrated the anti-inflammatory activity of Vitexdoniana leaves, as well as their mechanism of action.The phytochemical analysis of the extract (Vitexdoniana) revealed the presence of flavonoids, glycosides, tannins and saponins. Some isolated flavonoids (quercetin, wogonin, nevadensin, and quercetinpentamethyl ether) possess strong anti- inflammatory activities (Reinhart, 1955). Biflavonoids in particular show advantages over certain classical non-steroidal anti-inflammatory drugs. Such advantages include high margin of safety and least ulcerogenicity (Rageeb and Barhate, 2011). This implies that flavonoids with non-

steroidal anti-inflammatory activity might decrease the risk of gastrointestinal damage (Palmer and Gosh, 1981).

The anti -inflammatory activity of the bioflavonoids of Gareinia kola have been demonstrated by Igboko (1987). Others are Azadirachtaindica (Winter et al., 1963; Okpanyi and Ezeukwu, 1981), Dashanasamskarachurna (Peiriset al., 2011) Cissusquadrangularis (Priyanka and Rekha, 2010). There is increased advocacy for the consumption of anti-inflammatory foods including fruits such as cucumber, vegetables, certain nuts for example coconut and some spices like onion (Hyman and Mark, 2006). Reports have shown that these plants have high chemical and nutrient profile such as vitamins, fats, oil, alkaloids, retinoids, bioflavonoids, tannins, saponins, and antioxidants some of which possess anti-inflammatory activity (Hyman and Mark, 2006).

There is evidence for both oxygen-centred free radical and products of complement activation acting as mediators of inflammation, and the generation and reaction of free radicals at sites of inflammation in several inflammatory conditions (Arora et al., 2000). Antioxidants in these plants scavenge these free radicals and thus exert anti-inflammatory properties. The antioxidant chemicals found in many fruits and vegetables are the main benefits of high intake of these foods in the diet. These antioxidants scavenge excess free radicals produced during inflammation and also prevent the free radicals from oxidizing sensitive biological molecules and thus reduce the incidence of diseases (Cho et al., 2005).

1.10Phytochemistry

Phytochemistry is the study of phytochemicals. Phytochemicals are secondary metabolites produced by plants.They occur in various parts of a plant. Their functions are diverse and include provision of strength to plants, attraction of insects for pollination and feeding, while some are simply waste products (Ibegbulemet al., 2003).They give plants colour, flavour, smell and are part of a plant’s natural defence system (Agatemor et al., 2009; Ejele and Akujobi, 2011). These compounds have been linked to human health by contributingto protection against degenerative diseases (Dandjessoet al., 2012). Phytochemicals are present in varieties of plants utilized as important components of both human and animal diets. These include fruits, seeds, herbs and vegetables (Okwu, 2005). Different mechanisms have been suggested for the action of phytochemicals.  They  may  act  as  antioxidants,  or  modulate  gene  expression  and  signal

transduction pathways  (Dandjessoet  al.,  2012).  They  may  be  used  as  chemotherapeutic  or chemopreventive agents (Paolo et al., 1991).

Phytochemicals are formed during the plant normal metabolic processes. These chemicals are often  referred  to  as  “secondary  metabolities”  of  which  there  are  several  classes  including alkaloids, flavonoids, coumarins, glycosides, gums, polysaccharides, phenols, tannins, terpenes and terpenoids (Harborne, 1973; Okwu, 2005). Phytochemicals are naturally occurring and are believed to be effective in combating or preventing disease due to their antioxidant properties (Ejeleet al., 2012). The medicinal values of these plants lie in their constituent phytochemicals, which produce the definite physiological actions on human body. The most important of these phytochemicals are alkaloids, tannins, flavonoids and phenolic compounds (Iwu, 2000).Some of these naturally occurring phytochemicals are anti-carcinogenic and some others possess other beneficial properties, and  are referred to  as chemopreventers. Among the  most  investigated chemopreventers are  some  vitamins,  plant  polyphenols,  and  pigments  such  as  carotenoids, chlorophylls, flavonoids, and betalains (Ejeleet al., 2012).

1.10.1 Tannins

Tannins  are  an  exceptional group of water  soluble  phenolic  metabolites  of relatively  high molecular weight and having the ability to complex strongly with carbohydrates and proteins (Heldt and Heldt, 2005). Tannins are astringent, bitter plant polyphenols and the astringency from tannins is what causes the dry and pucker feeling in the mouth following the consumption of unripened fruit or red wine (Serafiniet al., 1994). They are grouped into two forms, hydrolysable and condensed tannins (Nityanand, 1997). Hydrolysable tannins are potentially toxic and cause poisoning if large amounts of tannin-containing plant materials such as leaves of oak (Quercusspp.) and yellow wood (Terminalia oblongata) are consumed (Heldt and Heldt, 2005) and as such seen as one of the anti-nutrients of plant  origin  because of their capability to precipitate proteins, inhibit the digestive enzymes and decline the absorption of vitamins and minerals (Khattabet al., 2010).

Several health benefits have been attributed to tannins and some epidemiological associations with decreased frequency of chronic diseases have  been established (Serrano  et  al.,  2009). Several studies have shown significant biological effects of tannins such as antioxidant or free

radical scavenging activity as well as inhibition of lipid peroxidation and lipoxygenasesin-vitro (Amarowiczet al., 2000). They have also been shown to possess anti-microbial, anti-viral anti- mutagenic and anti-diabetic properties (Gafneret al., 1997). The antioxidant activity of tannins results from their free radical and reactive oxygen species-scavenging properties, as well as the chelation of transition metal ions that modify the oxidation process (Serrano et al., 2009).

1.10.2 Phenols

Phytochemicals such as phenolics, which are present in foods have attracted a great of attention (Agatemor et al., 2009). Phenols sometimes called phenolicsarea family of organic compounds characterized by a hydroxyl (-OH) group attached to a carbon atom that is part of an aromatic ring. Besides serving as the generic name for the entire family, the term phenol is also the specific name for its simplest member, monohydroxybenzene (C6H5OH), also known as benzenol or carbolic acid (Amorati and Valgimigli, 2012). They also are produced by plants and microorganisms, with variation between and within species. Organisms that synthesize phenolic compounds do so in response to ecological pressures such as pathogen and insect attack, UV radiation and wounding(Mishra and Tiwari, 2011). The largest and best studied natural phenols are  the  flavonoids, which  include several thousand compounds, among them the  flavonols, flavones, flavan-3ol, flavanones, anthocyanidins and isoflavonoids.Phenolics as secondary metabolites  are  present  in  plants  and  contribute  to  the  development  of  colour,  taste  and palatability as well as the defence system of plants (Agatemor et al., 2009).

1.10.3Flavonoids

Flavonoids area large family of polyphenolic compounds mainly of plant origin, ubiquitous in nature and are categorized according to their chemical structures into flavones, anthocyanidins, isoflavones, catechins, flavonols, chalcones and flavanones (Robak and Gryglewski, 1988). They occur mostly in vegetables, fruits and beverages like tea, coffee and fruit drinks. They accumulate in plants as phytoalexins defending them against microbial attack (Harborne, 1973); and fungal attack (Oloyedeet al., 2010).

Flavonoids have been found to possess many useful effects on human health. They have been shown to have several biological properties including anti-inflammatory activity, enzyme inhibition, antimicrobial activity, oestrogenic activity (Malairajanet al., 2006; Atanassovaet al.,

2011), antioxidant and free-radical-scavenging ability (Cook and Shamman, 1996). Flavonoids have also been shown to exhibit anti-leukemic properties and mild vasodilatory properties useful for the treatment of heart disease (Odugbemiet al., 2007).

1.10.4Anthocyanins

Anthocyanins are water-soluble vascular pigments that may appear red, purple, or blue depending on the pH.Anthocyanins occur in all tissues of higher plants, including leaves, stems, roots, flowers, and fruits (Andersen, 2001).In flowers, bright-reds and -purples of anthocyanins are adaptive for attracting pollinators. In fruits, the colourful skins also  attract  the attention of animals, which may eat the fruits and disperse the seeds. In photosynthetic tissues (such as leaves and sometimes stems), anthocyanins have been shown to act as a “sunscreen”, protecting cells from high-light damage by absorbing blue-green and ultraviolet light, thereby protecting the tissues from photoinhibition, or high-light stress (Jack, 1998).In addition to their role as light- attenuators, anthocyanins also act as powerful antioxidants. However, it is not clear whether anthocyanins can significantly contribute to scavenging of free radicals produced through metabolic processes in leaves, since they are located in the vacuole and, thus spatially separated from metabolic reactive oxygen species. Some studies have shown hydrogen peroxide produced in other organelles can be neutralized by vascular anthocyanin.It may protect the leaves from attacks by plant eaters that may be attracted by green colour (Karageorgou and Manetas, 2006).

1.10.5 Alkaloids

Alkaloids play a very important role in organism metabolism and functional activity. They are metabolic products in plants, animals and micro-organisms. They occur in both vertebrates and invertebrates as endogenous and exogenous compounds. Many of them have a disturbing effect on the nervous systems of animals. Alkaloids are the oldest successfully used drugs throughout thehistorical treatment of many diseases (Aladesanmiet al., 1998) and are one of the most diverse groups of secondary metabolite found in living organism. They have an array of structural types, biosynthetic pathways, and pharmacological activities (Tankoet al., 2008). In plants and insects, toxic alkaloids are sequestered for use as a passive defence mechanism by acting as deterrents for predating insects (Eyonget al., 2006).However, they inhibit certain mammalian enzyme activities

such as those of phosphodiesterase, thus prolonging the action of cyclic AMP. At concentrations of these alkaloids in edible plants, they are usually non-toxic (Okakaet al., 1992).

Alkaloids have been used throughout history in folk medicine in different regions of the world. They have been a constituent part of plants used in phytotherapy. Many of the plants that contain alkaloids are just medicinal plantsand have been used as herbs. Some alkaloids that have played an important role in this sense include aconitine, atropine, colchicine, coniine, ephedrine, ergotamine, mescaline, morphine,strychnine, psilocin and psilocybin (Aladesanmiet al., 1998).

Many alkaloids are known to have effect on the central nervous system. Some alkaloids act as antiparasitic (such as morphine, a pain killer). For example, quinine was widely used against Plasmodium falciparum. In this respect, it is found from the phytochemical screening that most plants traditionally used to treat malaria contain alkaloids among other things (Jerutoet al., 2011).

1.10.6 Glycosides

Glycosides play numerous important roles in living organisms. In plants, chemicals are stored in the form of inactive glycosides. These can be activated by enzyme hydrolysis (Brito-Arias,

2007), which causes the sugar part to be broken off, making the chemical available for use. Many such plant glycosides are used as medications. In animals and humans, poisons are often bound to sugar molecules as part of their elimination from the body.Glycosides can be classified by the glycone, by the type of glycosidic bond, and by the aglycone. By aglycone, glycosides are classified as anthraquinone glycosides, coumarin glycosides, cyanogenic glycosides, etc. Although glycosides form a natural group in that they contain a sugar unit, the aglycones are of such varied nature and complexity that glycosides vary very much in their physical and chemical properties and in their pharmacological action (Trease and Evans, 2002). From ancient times, humans have used cardiac-glycoside-containing plants and their crude extracts as arrow, ordeal, homicidal, suicidal and rat poisons, heart tonics, diuretics and emetics. In modern times, purified extracts or synthetic analogues of a few have been adapted for the treatment of congestive heart failure and cardiac arrhythmia.

1.10.7 Sterols

Sterols  are  triterpenes which  are  based  on  the  cyclopentanehydrophenanthrene ring  system (Harborne, 1973). Sterols in plants are generally described as phytosterols with three known types occurring in higher plants: sitosterol (formerly known as β-sitosterol), stigmasterol and campsterol(Harborne, 1973). These common sterols occur both as free and as simple glucosides. Sterols have essential functions in all eukaryotes. Free sterols are integral components of the membrane lipid bilayer where they play important role in the regulation of membrane fluidity and permeability (Irvine, 1961). While cholesterol is the major sterol in animals, a mixture of various sterols is present in higher plants, with sitosterol usually predominating. However, certain sterols are confined to lower plants such as ergosterol found in yeast and many fungi while others like fucoterol, the main steroid of many brown algae is also detected in coconut (Harborne, 1973).

1.10.8 Resins

Chemically, resins are complex mixtures of resin acids, resins alcohols (resinols), resin phenols (resinotannols), esters and  chemically  inert  compounds known as  resenes.  Resins  are  often associated with volatile oils (oleoresins), with gums (gum-resins) or with oil and gum (oleo-gum- resins).The resin produced by most plants is a viscous liquid, composed mainly of volatile fluid terpenes, with lesser components of dissolved non-volatile solids which make resin thick and sticky (Trease and Evans, 2002). The most common terpenes in resin are the bicyclic terpenes alpha-pinene, beta-pinene, delta-3 carene and sabinene, the monocyclic terpenes limonene and terpinolene, and smaller amounts of the tricyclic sesquiterpenes, longifolene, caryophyllene and delta-cadinene.

1.10.9 Terpenoids

Terpenoids, also known as isoprenoids are the major family of natural compounds, comprising more  than  40,000  different  molecules.  The  isoprenoid  biosynthetic  pathway  produces both primary and secondary metabolites that are of great significance to plant growth and persistence (Trease and Evans, 2002). Terpenoids are secondary metabolites that have molecular structures comprising carbon backbones that are made up of isoprene (2-methylbuta- 1, 3-diene) units. The terpenoids are comprised of two isoprene units, containing ten carbon atoms. Among the primary metabolites produced by this pathway are: the phytohormones-abscisic acid (ABA); gibberellic acid  (GAs)  and  cytokinins;  the  carotenoids;  plastoquinones  and  chlorophylls  involved  in

photosynthesis; the ubiquinones required for respiration; and the sterols that impact membrane structure (Harborne, 1973). Many of the terpenoids are important for the quality of agricultural products such as the flavour of fruits and the fragrance of flowers like linalool (Singh, 2009). In addition, terpenoids can have  medicinal properties such as anti-carcinogenic (e.g. taxol and perilla alcohol), antimalarial (e.g. artemisinin), anti-ulcer, antimicrobial or diuretic (e.g. glycyrrhizin) activity (Harrawijnet al., 2001). The steroids and sterols in animals are biologically produced  from precursors of terpenoid  and  sometimes terpenoids are  added  to  proteins  to increase their attachment to the cell membrane, a process known as isoprenylation (Singh, 2009).

1.10.10 Saponins

Saponins are groups of secondary metabolites found widely distributed in the plant kingdom as plant glycosides, characterized by a skeleton of 30-carbon precursor oxidosqualene to which glycosyl residues are attached along with it, they have sturdy foaming property. (Harborne,

1973). They are subdivided into triterpenoids and steroid glycosides and are stored in plant cells as inactive precursors but  are readily converted into  biologically active antibiotics by plant enzymes in reply to pathogenic attack (Okwu, 2005).

Saponins  protect  plants  against  attack  by  pathogens  and  pets  (Jerutoet  al.,  2011).  These molecules also have substantial marketable value and are processed as drugs and medicines, foaming agents, sweeteners, taste converters and cosmetics (Kensil, 1996).They have the ability to haemolyse red blood cells and confer a bitter taste to fruits. Saponin containing plants are used as traditional medicines, especially in Asia, and are intensively used in food, veterinary and medical industries (Kensil,1996). The pesticidal activity of saponins has long been reported (Irvine,  1961).  Saponin-glycosides  are  very  lethal  to  cold-blooded  organisms,  but  not  to mammals (Kensil,1996). Plant extracts containing a high percentage of saponins are commonly used  in  Africa  to  treat  water  supplies  and  wells  contaminated  with  disease  vectors;  after treatment, the water is safe for human drinking (Kensil,1996). Saponins induce a strong adjuvant

effect to T-dependent as well as T-independent antigens and also induce strong cytotoxic CD8+

lymphocyte responses and potentiate the response to mucosal antigens (Kensil, 1996). They have both stimulatory effects  on the  components of specific  immunity and  non-specific immune reactions such as inflammation (Chukwujekuet al., 2005) and monocyte proliferation (Aggarwal and Shishodia, 2006).

Saponins have long been known to possess lytic action on erythrocyte cell membranes and this property has been used in their detection. The haemolytic actions of saponins are alleged to be due to their affinity for the aglycone moiety of membrane sterols, mainly cholesterol with which they form undissolvable complexes (Davies, 1995).

1.10.11 Reducing sugar

A sugar is classified as a reducing sugar only if it has an open-chain form with an aldehyde group or a free hemiketal group. A reducing sugar is one that reduces certain chemicals. Sugars with ketone groups in their open chain form are capable of isomerizing via a series of tautomeric shifts  to  produce  an  aldehyde  group  in  solution  (Campbell  and  Farrell,  2012).  That  is, saccharides bearing anomeric carbons that have not formed glycosides are termed reducing sugars, because the free aldehyde group that is in equilibrium with the cyclic form of the sugar reduces mild oxidizing agents. Identification of a sugar as non-reducing is an evidence that it is glycoside (Voet et al., 2013).

1.11Hepatotoxicity

Hepatotoxicity implies chemical-driven liver damage. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Other chemical agents, such as those used in laboratories (e.g. CCl4, paracetamol) and industries (e.g.  lead,  arsenic),  natural  chemicals  (e.g.  microcystins,  aflatoxins)  and  herbal  remedies (cascara, sagrada, ephedra) can also induce hepatotoxicity (Singhet al., 2012). Chemicals that cause liver injury are called hepatotoxins.  These agents are converted into chemically reactive metabolites in liver, which have the ability to interconnect with cellular macromolecules such as protein, lipids and nucleic acids, leading to protein dysfunction, lipid peroxidation, DNA damage and oxidative stress. This damage of cellular function can dismiss in cell death and likely liver failure. More than 900 drugs have been implicated in causing liver injury and it is the most common reason for a drug to be withdrawn from the market. Chemicals often cause subclinical injury to liver which manifests only as abnormal liver enzyme tests. Drug-induced liver injury is responsible for 5% of all hospital admissions and 50% of all acute liver failures. More than 75 percent of cases of idiosyncratic drug reactions result in liver transplantation or death (Ostapowiczet al.,2002).

1.11.1 The liver

The liver plays a pivotal role in regulating various physiological processes. It is also involved in several vital function, such as metabolism, secretion and storage. It has great capacity to detoxify toxic  substances and  synthesize  useful principle (Domitrovic et  al.,  2013).  It  helps  in  the maintenance, performance and regulating homeostasis of the body. It is involved in almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction. It aids metabolism of carbohydrate, protein and fat, detoxification, secretion of bile and storage of vitamins (Ahsan et al., 2009). The role played by the organ in the removal of substances from the portal circulation makes it  susceptible to first and persistent attack by offending foreign compounds, culminating in liver dysfunction. These hepatotoxic agents activated some enzyme activities in the cytochrome P-450 system such as CYP2E1 leading to oxidative stress (Singhet al., 2012). Injury to hepatocyte and bile duct cells lead to accumulation of bile acid inside liver. This promotes further liver damage.

The liver is also the major reticula endothelial organ in the body as such has important immune function in maintaining body veracity. Damaging hepatocyte results in the activation of innate immune  system  like  kupffer  cells  (kc),  natural killer  cells  (NK)  and  natural killer  T-cells (NKT)and result in producing pro inflammatory mediators such as tumour necrosis factor-α (TNF), interferon-γ (IFN), and interleukin-β (IL) produced liver injury. Many agents which damage an intracellular organellemitochondria include drug accumulation, inhibition of electron transport  and  fatty acid  oxidation or  depletion of anti-oxidant  defences.  An  indirect  result ensuing from mitochondrial participation in programmes of cell death. These programmes lead to necrosis or apoptosis, they are mediated through signalling mechanisms arising at the cell membrane (e.g. death receptors) or in subcellular compartments (e.g. the endoplasmic reticulum or cell nucleus) (Sun et al., 2001; Friedman, 2000). Its dysfunction releases excessive amounts of oxidants which, in turn injure hepatic cells. Non-parenchymal cells such as kupffer cells, fat storing stellate cells, and leucocytes (i.e. neutrophils and monocytes) also and in the mechanism of hepatotoxicity (Patel et al., 1998). Hepatic injury leads to disturbances in transport function of

hepatocytes resulting in leakage of plasma membrane thereby causing an increased enzyme level in the serum (Ibid).

1.11.2 Assay associated with hepatotoxicity

When the integrity of the membrane of the hepatocytes is compromised, certain enzymes located in the cytosol are released into the blood. Their estimation in the serum is useful quantitative marker for the evaluation of liver damage (Pari and Kumar, 2002). Glutamate dehydrogenase activity is not found in normal serum but moderate elevation is found in most cases of acute hepatitis indicating cellular damage. Another demonstrable type of membrane damage involves injury to lysosomes which leads to the release of acid ribonuclease, acid phosphatases, and other liver enzymes such alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), into the blood stream. These enzymes are elevated to distinguish and assess the extent and type of hepatocellular injury (Pari and Kumar, 2002). Other indicators used in hepatotoxicity studies are total bilirubin concentration, cholesterol concentration, low density lipoprotein (LDL) concentration, high density lipoprotein (HDL) and triacylglycerols concentrations.

1.12Carbon tetrachloride (CCl4)

Carbon tetrachloride (CCl4) was the formerly used for metal degreasing and as a dry-cleaning fluid, fabric-spotting fluid, fire extinguisher, grain fumigant and reaction medium. CCl4 -induced liver damage has been lengthily used as an experimental model. It is used as a model drug for the study of hepatotoxicity in acute and chronic liver failure. (Weber et al., 2003;Singhet al., 2012). CCl4  is metabolized by CYP2E1, CYP2β and possibly CYP3A to form the tri-chloromethyl radical, CCl3  (Poli, 1993). This CCl3 radical can bind to cellular molecules damaging crucial cellular progression. This radical can also react with oxygen to form the tri-chloromethyl peroxy radical CCl3OO, a highly reactive species. The metabolites of CCl4 cause the hepatic injury in the CCl4 liver injury model. Single dose of CCl4 to a rat produces centrilobular necrosis and fatty changes. The poison reaches its maximum concentration in the liver in 3hrs of administration

(Dawkins, 1963). Non-lethal intoxication triggers liver tissue remodelling and healing through the activation of hepatic stellate cells (HSCs), leading to liver fibrosis (Friedman, 2000). Tri- chloromethyl free  radical  is  believed  also  to  initiate  the  biochemical processes  leading  to oxidative stress, a direct cause of many pathological conditions such as diabetes mellitus, cancer, hypertension, kidney damage, liver damage and death. Liver damage caused by acute exposure to CCl4 shows clinical symptoms such as jaundice, swollen and tender liver and elevated levels of the liver enzymes -ALT, AST and ALP in the blood (Tirkeyet al., 2005).

1.13Cucumis Sativus(Cucumber)

1.13.1 Morphology

Cucumis sativus (Cucumber) is a widely cultivated plant in the gourd family of Cucurbitaceae, which also includes important crops such as melon, water melon, and squash. It is a creeping vine that roots in the ground and grows up trellises or other supporting frames, wrapping around supports with thin, spiralling tendrils. The plant has a large leaves that form a canopy over the fruit. The fruit of the cucumber is roughly cylindrical, elongated with tapered ends, and may be as large as 60 centimetres (24 inches) long and 10 centimetres (3.9 inches) in diameter. Having an enclosed seed and developing from flowers, botanically speaking, cucumber can be classified as an accessory fruits.

Figure 4: Cucumis sativus Fruits: Cucumber

Source: Prohens and Fernando, (2008).

1.13.2 Taxonomy and Nomenclature

Kingdom        –          Plantae Unranked        –          Angiosperms Unranked        –          Eudicots Unranked        –          Rosids

Order              –          Cucurbitales Family            –          Cucurbitaceae Genus             –          Cucumis Species           –          Cucumissativus Source: William and Brigitta, (2010)

1.13.3Nutritional composition

There is increased consumption of Cucumis sativus fruits possibly because of their high nutritional value. The nutritional compositionof Cucumis sativus include protein, fat, and carbohydrate as primary metabolites; and dietary fibre which is important for the digestive system. Cucumis sativus contains some essential vitamins and anti-oxidants which are effective in human health (Grubben and Denton, 2004; Wang et al.,2007). Table 1 shows some basic nutritional composition of a cucumber fruit.

Table 1.Nutritional composition of 100g edible portion of cucumber fruit.

Compound	Amount
Water	95.23%
Energy	42kJ
Protein	0.65g
Fat	0.1g
Sugars	1.5g
Dietary Fibre	0.5g
Starch	0.83g
Ca	16mg
Mg	8mg
P	24mg
Fe	0.28mg
Zn	0.1mg
Mn	0.079mg
K	147mg
Na	2mg

F	1.3μg
Carotene	60mg
Thiamin	0.027mg
Folate	7μg
Ascorbic acid	2mg
Pantothenic acid	0.259mg
Vitamin B6	0.04mg
Vitamin K	16.4μg
Vitamin A	105 IU
Lutein + Zeaxanthin	23ug

Source: National Nutrient Database for Standard Reference, USDA

1.13.4.Uses of Cucumis Sativus

(a) In Medicine: Cucumber (Cucumis sativus) is used by native people to cure many illnesses in some countries. In Africa, ripe raw cucumber fruits are used as a cure for sprue, a disease that causes flattering of the villi and inflammation of the lining of the small intestine; and in Indo China, cooked immature fruits are used to treat dysentery in children (Grubben and Denton,

2004). It  is also useful in fighting constipation, as the fibre content helps to overcome the hypotony which is the cause of constipation (Yohanna, 2013). Swapnilet al.(2012) reported the use of Cucumis Sativus in the treatment of patients with high blood pressure and with irritated skin as a result of sun burn.

(b) Food: As a fresh market vegetable in Europe, United States and many parts of the world, cucumber is mainly used in salads, but young and ripe fruits are used as cooked vegetables (Grubben and Dentons, 2004). It is a good health food for the diabetics (Sharminet al., 2013).

(c) In Cosmetics:Cucumis sativus-derived ingredients are reported to function in cosmetics as skin  conditioning  agents  (Gottschalck  and  Breslawec,  2012).  Products  containing  Cucumis sativus fruit extract are reported to be used on baby skin and may be applied to eye area or mucous  membrane.  Additionally,  Cucumis  sativus  fruit  extract  is  used  in  cosmetic  spray products for face, neck, body and hand. (Rotheet al., 2011).

1.14Rationale for study

It is believed that Cucumis sativusfruit has anti-oxidant activity, high flavonoid content, anti- inflammatory and analgesic effect (Kumar et al., 2010; Singh-Gill et al., 2010; Agarwal et al.,

2012). It is therefore necessary to establish some of these properties and their application in management of inflammation and liver diseases.

1.15Aim of study

The aim of this research is to assess the effect of the homogenate of Cucumis sativus fruit on some inflammatory models and CCl4-induced hepatotoxicity in rats so as to know the possibility of its implication in the management of diseases.

1.16Research objectives

This research work is set out to achieve the following specific objectives:

  To determine the phytochemical constituents of the homogenate of Cucumis sativus fruit.

  To determine the proximate composition of the homogenate of Cucumis sativus fruit.

  To determine the acute toxicityof thehomogenate of Cucumis sativus fruit.

  To  determine  effect  of the  homogenate  of  Cucumis sativus  fruit  on  DPPH  radical scavenging activity.

  To determine the anti-inflammatory effects of the homogenate of Cucumis sativusfruit on agar-induced paw oedema in rats.

  To determine the effect of the homogenate of Cucumis sativus fruit on hypotonicity- induced haemolysis of red blood cell.

  To determine the effect of the homogenate of Cucumis sativus fruit on phospholipase A2 activity.

  To determine the effect of the homogenate of Cucumis sativus fruit on prostaglandin synthase activity.

  To determine the effects of the homogenate of Cucumis sativusfruit on some serum biochemical parameters such as liver marker enzymes and lipid profileof rats intoxicated with CCl4.

  To carry out histopathological examination of the liver organ implicated in this study.

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