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The Medicinal Component Of Cucumber And Anti-Microbial Of Cucumber
THE MEDICINAL COMPONENT OF CUCUMBER AND ANTI-MICROBIAL OF CUCUMBER
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.
1.2.1Acute 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
Source:(Levisonet al.,2008)
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
Source: (Marieb and Mitchell, 2007)
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).
1.3.4Resolution
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.
1.6Antioxidants
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.
1.7Inflammatory 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.
1.8Anti-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.
Figure 3: Synthesis of prostaglandins
Source: (Koch et al., 2002)
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 antiulcerogenic 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 antiinflammatory 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 nonsteroidal 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 antimutagenic 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 lightattenuators, 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-gumresins).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). Trichloromethyl 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
- 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.
- 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).
- 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, antiinflammatory 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 hypotonicityinduced 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.