ANTIULCEROGENIC AND IMMUNOSTIMULATORY PROPERTIES OF THE AQUEOUS EXTRACT OF THE LEAVES OF Ficus capensis IN WISTAR ALBINO RATS

ABSTRACT

Ficuscapensis leaves have always played a great role in preventing, controlling and alleviating various disease conditions. This study was aimed at validating the traditional use of the leaves of Ficuscapensisin folk medicine as an anti- ulcer and immunostimulatory agent. Diclofenac sodium (100mg/kg) body weight was administered intraperitoneally to induce ulcer. Seven groups(five rats each)were used. Groups 2(150mg ranithidine),4,5,6and7 served as the ulcer test groups while group1 and 3 served as ulcer untreated group(negative and positive controls respectively).After 24 hour fasting, all the rats were sacrificed and the stomach was removed for observation of ulcer score, ulcer index and ulcer indices(SOD,CAT, MDA and GSH).Five groups(five rats each) were used to investigate delayed type hypersensitivity(DTH),humoral antibody(HA)and Myelosuppression. Groups 2,3, 4 and 5 (Levamisol) served as the test groups while group1 was the negative control.DTH in rats was sensitized by subcutaneous (SC) injection of 0.1ml of 10⁹ cells /ml sheep red blood cells (SRBCs) (day 0) in the planter region of the right hind foot paw and was challenged on day 5 by SC injection of the same concentration of antigen into the left hind paw. The oedema produced by antigenic challenge in the left hind paw was measured with a micrometer screw guage. HA in rats was immunized by ip injection of 0.2ml of 10⁹SRBCs/ml on day 0 and was challenged by injecting the same concentration on day 7.Primary antibody titre was determined on day 7(before the challenge) and the secondary titre was determined on day 14 by the haemagglutination technique. The highest dilution showing visible haemagglutination was taken as the antibody titre. Cyclophosphamide (30 mg/kg, i.p.) was used to induce myelo suppression and levamisole (50 mg/kg)was used as standard immune stimulating agents. After five days, blood sample was collected using rectobulba plexus in the eye. The before induction, after induction and after treatment blood samples obtained were used to determine the haematological parameters(WBC,Hb, PCV, RBC).The qualitative and quantitative phytochemical analysis showed  that the aqueous extract contained: reducing sugar(543.47 ± 0.004), saponins (2.39 ± 0.0032), tannin(7.57± 0.0015), flavonoid (1.27 ±  0.0321), soluble carbohydrates(3.92 ± 0.0030), alkaloid(3.34 ± 0.0015), steroid(1.04 ± 0.0321), hydrogen cyanide(1.61 ± 0.0026), glycoside(2.37 ± 0.0025), terpenoid (0.89  ± 0.0025),fats and oil.The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. The ulcer index decreased significantly (p<0.05) in the treatment groups compared to positive control (group 3).MDA activity decreases significantly(p<0.05) compared to group 3. The antioxidant enzymes: CAT,GSH and SOD activities of the test groups significantly increased (p <0.05) compared to group 3.DTH SRBC challenged rats showed a comparable increase in thickness of rat footpad 24hr and 48hr after challenge. Administration of extract produced significant increase (p<0.05) in footpad swelling of the rats compared to control. Significant increase (p<0.05) in the primary and secondary SRBCs – specific mean antibody titre values after treatment was observed compared to control. Cyclophosphamide caused a significant (p<0.05) decrease in haematological parameters. Treatment with extract or levamisol resulted in a restoration of the bone marrow activity. Haematological parameters of the test groups significantly increased (p <0.05) compared to control. The findings indicated that the extract could be used in the management of ulcer and immunosuppressive disorders.

CHAPTER ONE

INTRODUCTION

            Plant materials are sources of shelter, food and medicinal compounds which have been known to play a dominant role in the maintenance of human health in most rural communities in the developing countries (Oduola et al., 2005). Herbal medicine is fast emerging as an alternative treatment to available synthetic drugs for the treatment of diseases possibly due to lower cost, availability, fewer adverse effects and perceived effectiveness (Ubaka et al., 2010). The World Health Organisation (WHO) has shown great interest in plant derived medicines which have been described in the folklore medicines of many countries (Mukheerjee, 2002). However, the historic role of medicinal plants in the treatment and prevention of diseases and their role as catalysts in the development of pharmacology do not however, assure their safety for uncontrolled use by an uninformed public (Matthews et al., 1999). In Enugu State of Nigeria, many plants are used in herbal medicine to treat diseases, as well as to promote rapid healing of wound and sores (Dimo et al., 2002).

 

1.1 Ulcer

An inflammatory, usually suppurating, lesion on the skin or internal mucous surface that results in necrosis is referred to as ulcer (Nwodo, 2012).  A peptic ulcer also known as ulcer pepticum or peptic ulcer disease (PUD) is defined as a disruption of the mucosal integrity of stomach, duodenum or oesophagus leading to a local defect or excavation due to active inflammation (Adreoli et al., 2008).

• Brief history of peptic ulcer diseases

The high acidity (pH -2) or hyperchlorhydria was then believed to cause erosion of the stomach lining in the absence of foul; thus causing the development of the gastric ulcers (Toohey, 1974). The existence of gastric ulceration was acknowledged by Dicotes of Cargstos 350 BC (Gulia and Choudhary, 2011). In 1983, Warren and Marshall provided the first insight into an important pathogenic factor in peptic ulcer disease with the discovery of Helicobacter pylori (H. pylori). The aetiopathogenesis of peptic ulcer has changed from Schwartz’s dictum “No-acid-No ulcer” to “No Helicobacter pylori-No ulcer” (Shankaran and Dasai, 1995). Buckley and O’ Morain (1998) hypothesized that ulcers are caused by bacteria and chemical factors respectively. Reduction of gastric acid production as well as re-enforcement of gastric mucosal production has been the major approaches to cure peptic ulcer disease.Peptic ulcer disease refers to painful sores or ulcers in the lining of the stomach or first part of the small intestine, called the duodenum which impairs the quality of life and is associated with increased morbidity and mortality (Gopinathans and Rameela, 2014).

 

1.1.2 Pathogenesis of peptic ulcer

Considering the etiology of peptic ulcers, there are two prime factors responsible: the “aggressive” factors (gastric acid, pepsin, free radicals, infectious agents like Helicobacter pylori, chemicals and to a lesser extent bile salts and pancreatic enzymes) and the “defensive” factors (the adherent gastric mucus, bicarbonate, prostaglandins and mucosa blood flow. Ulcers are caused as a result of imbalance between aggressive and defensive factors.  Due to any cause, an increase in aggressive factors or a decrease in defensive factors will lead to loss of mucosal integrity resulting in ulceration (Gopinathan and Rameela,2014).A typical example is observed when mucosal defenses fail; resulting in a drop in the mucosal blood flow, delay in gastric emptying and epithelial restitution impairment.

Fig. 1: Role of aggressive and defensive factors in the pathogenesis of peptic ulcer (Source: Gulia and Choudhary, 2011).

• Factors that produce ulceration.

There are many factors that produce ulceration by aggravating gastric acid and pepsin secretion. These are discussed below.

1.2.1Endogenous factors producing ulceration

These include different visceral neurotransmitters and hormones which include the acetylcholine, gastrin, histamine, somatostatin and cholecystokinin, the second messengers (Ca²⁺) and the genetic factors.

1.2.1.1 Acetylcholine (ACh)

Acetylcholine causes gastric acid secretion in gastric phase of acid secretion (Matsuno et al., 1997).Acetylcholineexerts a considerable influence on gastric acid secretion through the direct stimulation of parietal cells, the release of gastrin from the pyloric antrum, and modifies the responsiveness of the parietal cells to gastrin and histamine (Bunce et al., 1977). It is released from vagus nerve and parasympathetic ganglion cells, which are located in gastric mucosa and stimulates acid secretion directly by gastrin release and indirectly by stimulating release of histamine (Walker, 2002).  It also stimulates the secretion of pepsinogen, the inactive precursor of pepsin, which also causes peptic ulcer (Venables, 1986).

1.2.1.2 Gastrin

Gastrin is an important hormone that plays a central role in the regulation of gastric acid secretion. This is demonstrated by the marked reduction in acid secretion produced by administering gastrin receptor antagonists. The hormone is produced by the G-cells, which are situated within the glands present in the antral region of the parietal cells found in different regions of the stomach. Gastrin stimulates acid secretion not by acting directly on the parietal cells but by acting on the enterochroma n-like (ECL) cells, which are situated in close proximity to the parietal cells (Code, 1956).These ECL cells have gastrin receptors that cause the cell to release histamine when activated by gastrin. The histamine released in this way then acts on histamine-2 receptors on the adjacent parietal cells causing them to secrete acid.The trophic effect of gastrin is not conned to the ECL cells but also affects the parietal cells and surface epithelial cells of the mucosa (Miyazaki et al., 1999).  The release of gastrin by the G-cells in the antral mucosa is inhibited by low antral luminal pH (Peters et al., 1983).This serves as an important negative feedback control of gastric acid secretion. The acid inhibition of gastrin release is mainly mediated by the release of somatostatin from D-cells situated close to the antral G-cells.

Fig. 2: The role of gastrin in the regulation of acid secretion (Source: Harty et al., 1986)

1.2.1.3 Histamine

Histamine has a key role in the physiological control of gastric acid secretion, which was established with the advent of the histamine H₂-receptor antagonists which have in vivo been shown to inhibit virtually all forms of basal and stimulated secretion (Soll et al., 1980).The presence of histamine stores in the gastric mucosa has clearly been established, although the type of storage cell is species dependent, being predominantly the enterochrommafin-like cell in the rat (Soll et al., 1980). Potentiating interactions between histamine and the other secretory stimulants at the parietal cell level have led to the hypothesis that histamine acts in a permissive role, markedly amplifying the effect of gastrin and acetylcholine on parietal cell receptors. It is the removal of this permissive effect of histamine, which accounts for the inhibitory actions of H₂-receptor antagonists. It exerts its ulcerogenic effect during abnormal physicology by acting through H₂-receptors on parietal cell(Soll et al., 1980).

Fig. 3: Mechanism involved in regulation of gastric acid secretion by the parietal cells.

Key: R= Receptors, EP₃= PG receptor for PGE₂, M₁= Muscarinic receptor, ECL= Enterochromaffin like cells, Ach = Acetylcholine, H₂ = Histamine. (Source: Pahwa et al., 2010).

1.2.1.4 Somatostatin and Cholecystokinin

Amajor evidence that cholecystokinin (CCK) acts as inhibitor of gastric acid secretion is that exogenous CCK infused intravenously in a physiological dose is capable of inhibiting gastric acid secretion (Konturek et al., 1992). Both receptor subtypes CCK A and CCK B are involved in inhibition and facilitation of gastrin action respectively. On activation of CCK A receptor, somatostatin is released which act through somatostatin receptors on gastrin G- cells to inhibit gastrin secretion and CCK B receptor stimulation by CCK causes increased release gastric acid (Chayvialle et al., 1978). So, it can be assumed that peptic ulcer patients are deficient in response of CCK A receptor activation by endogenous CCK, resulting in deficiency of somatostatin and increased gastric acid release (Harty et al., 1986).

 

1.2.1.5 Calcium ion (Ca²⁺⁾ as second messengers

Ca²⁺ plays an important role in pathogenesis of gastric ulcers. An increase in cytosolic free calcium increases the effects of ACh and gastrin on stimulation of acid secretion by parietal cells (Kadalmani, 2011).Ca²⁺ also plays an important role in the release of histamine from enterochromaffin-like (ECL) cells, a powerful chemical mediator of gastric acid secretion, which involves both mobilization of an intracellular calcium pool and influx of calcium over the ECL- cell membrane (Sandvik et al., 2000).

1.2.1.6 Genetic factors

Increased familial history is found in 20-50% of patients. Ulcers are also more common in blood group O individuals and in those who do not secrete blood group antibodies into gastric secretions (Green and Harris, 2008).

1.2.2Exogenous factors producing ulceration

These include bacterial infection (H.pylori), non steroidal anti -inflammatory drugs (NSAIDs),alcohol,psychogenic factors and dietary habit.

1.2.2.1 Helicobacter pylori (H.pylori)

H.pylorus is a curved or S-shaped gram negative bacterium approximately 0.5 by 3 μm in size containing four to seven sheathed flagella at one pole. Gastric ulceration is the process of ulcer formation where there is a break in the tissues lining the stomach. Gastric ulcerations are mainly associated with the presence of the bacterium Helicobacter pylori; other factors could be genetic predisposition, drugs such as non steroidal anti -inflammatory drugs (NSAIDS), alcohol, diet and smoking. Helicobacter pylorus causes a chronic low-level inflammation of the stomach lining and is strongly linked to the development of duodenal and gastric ulcers and stomach cancer (Suerbaum and Machetti, 2002). More than 50% of the world’s population harbour H. pylori in their upper gastrointestinal tract (Yamaoka and Yoshio, 2008). It contains hydrogenase used to obtain energy and produces oxidase, catalase and urease. It is capable of forming biofilms and can convert from spiral to a possibly visible but non-cultural coccoid form both likely to favour its survival and can be factors in theepidemiology ofthe bacterium (Josenhans etal., 2000). H. pylori, through chemotaxis moves away from the acidic environment of the lumen towards the more neutral pH environment of the epithelial surface for easy adhesion to membrane associated lipids and carbohydrates (Kusters et al., 2006).

Transmission of H. pylori is through oral means of faecal matter through the ingestion of waste-tainted water. Person to person transmission is either by oral-oral or faecal- oral route is most likely as the bacterium can be isolated from saliva, faeces and plaque of some infected persons (Brown, 2000).The success of a pathogen depends on both its virulence and its pathogenicity.  Sufficient number of H. pylori must survive the gastric acid barrier and colonize the enteric fluid or mucous layer through attachment mechanisms and motility in the intestinal mucous layer. Once the organism is established in the gut, pathogenic effects on the host may be produced by one or several means; examples are physical effects, elaboration of enzymes or toxins, and competition with the host for nutrients.

1. pylori have cell wall associated lectins which allow them to bind selectively to mucus and epithelial cells. Targets of H. pylori lectins exist in the gastric mucus as glycoprotein and glycolipids.H.pylori appears to bind to all of these, including sulphated (acid) mucins, L-fructose, D-galactose, sialic acids. H. pylori lectins also attaches to red blood cells of various animal species (Emody et al., 1988).H.pylorus attaches tightly to the epithelial cell, and a characteristic structure called an ‘attachment pedestal’ forms. This attachment causes localized cell damage characterized by enhancement of microvilli and disruption of cytoskeletal elements of the cell (Goodwin et al., 1986).

The most common and virulence producing enzyme produced by H. pylori is urease. This enzyme is highly active between the pH of 5 and 8.The urease produced by H. pylori breaks down urea (which is normally secreted into the stomach) to carbon dioxide and ammonia and the ammonia is converted to ammonium by taking a proton (H⁺) from water, which leaves only a hydroxyl ion, producing bicarbonate which neutralizes gastric and this accounts for the survival of H. pylori in the acidic stomach (Smoot, 1997). It hydrolyses urea into ammonia. Ammonia thus generated acts as a potent cellular toxin in 3 ways (Megraud et al., 1992). Firstly, it combines with α-ketoglutarate to form glutamine, thus depleting Krebs cycle of an essential intermediate substrate (Nelson and Cox, 2004.). Secondly, it interacts with hypochlorus acid to form mono-n-chloramine, which also acts as potent cellular toxin (Graham et al., 1992). Thirdly, its toxic effect may also be mediated by neutrophil generated oxygen radicals because it is inhibited by anti-neutrophil serum (Suzuki et al., 1992). Thus, urease enzyme plays an important role in the virulence of H.pylori and its colonization in gastric mucosa. The release of free oxygen radicals by the neutrophils might play a role in the genesis of chronic inflammation and in the development of peptic ulcer (Salim, 1993).

1. pylori also produces certain chemotoxins, known asvacuolating cytotoxin (VacA) which directly act on epithelial cell surface and damage defence system ( Leunk et al.,1988). VacA renders the cell membrane permeable to urea by causing formation of transmembrane pores, suggesting it can increase H. pylori pathogenicity by enhancing urease activity (Tombola et al., 2001).

1.2.2.2 Non-Steroidal Anti-inflammatory Drugs (NSAIDS)

Non-steroidal anti-inflammatory drugs use has been associated with the development of gastric ulcers and with the major complications of ulcers i.e. gastrointestinal bleeding and perforation as side effects; they can even lead to death (Armstrong and Blower, 1987; Faulknera et al., 1988).  NSAIDs reduce tissue levels of prostaglandins, especially PGE₂, PGE₁ and PGI₂, by inhibiting cyclo-oxygenase-1(COX-1), which is the most important mechanism action. NSAID cause ulceration by generation of oxygen derived free radicals and products of the lipooxygenase pathway (Rainsford, 1989). COX inhibition by NSAID results in diversion of arachidonic acid metabolism towards lipooxygenase pathway, resulting in increased leukotriene synthesis. These leukotrienes can contribute to gastrointestinal ulceration, via two mechanisms. Firstly, by a reduction in prostaglandin levels of both PGE and PGF, that induces the secretion of mucin which acts as a protective agent against potential stomach ulceration induced by HCl and pepsin and secondly, through the release of oxygen radicals mediated mucosal injury produced in this pathway (Alrdahe et al., 2010).

Prostaglandins (PGs) have long been known to be mucoprotective and ulcer healing agents. Prostaglandins protect GI mucosaby forming a cytoprotective layer and increasing thesecretion of bicarbonate ions that neutralise the gastricacidity. All therapeutically useful NSAIDs act by inhibiting the synthesis of PGs. Cyclooxygenase has two isoforms,one constitutive (COX-1) and another inducible (COX-2).A third isoform (COX-3) has recently been described aswell. NSAIDs are now divided into selective (thoseinhibiting COX-2) and non-selective (inhibiting both COX-1 and COX-2). Conventional NSAIDs cause non-selectiveinhibition of cyclooxygenase, which leads to reduction inbicarbonate secretion and reduced mucous production.Coupled with it is vasoconstriction that occurs due toNSAIDs, which causes hypoxia and consequent formationof ulcer. Most NSAIDs are weak organic acids and havelow pKa. Therefore, they remain unionised in stomach andare absorbed appreciably from stomach(Devilin,2011). However, oncethey breach the cell membranes of stomach cells andreach within, they encounter a basic pH (e.g., 7.1). Thiscauses so called “trapping” of the drugs inside the cell.This topical effect is considered an important mechanismof gastro-duodenal damage associated with their use.Even short-term (< 1 week) use of aspirin and other nonsteroidalanti-inflammatory drugs (NSAIDs) can precipitate ulcer-related bleeding. Risk of ulcer development isincreased in patients with advanced age, positive familyhistory, female sex, prolonged use of high dose of NSAIDs and concomitant use of other gastrotoxic or anticoagulantdrugs, alcoholism, heavy coffee consumption, and poor general health.NSAID induced GI damage is of three main types;superficial damage such as mucosal haemorrhagesand erosions, endoscopically documented non-symptomatic(silent) ulcers and symptomatic ulcers causing complications such as GIhaemorrhage(Devilin,2011).

Fig. 4: Structure of Diclofenac-sodium

1.2.2.3 Ethanol

Ethanol damage to the gastrointestinal mucosa starts with micro-vascular injury, namely disruption of the vascular endothelium resulting in increased vascular permeability, oedema formation and epithelial lifting (Ohya and Guth, 1988).  Alcohol also causes the stomach cells to over secrete both acid and histamine which make the stomach linings vulnerable to ulcer formation. Ethanol also reduces prostaglandin levels, increases the release of histamine and influx of calcium ions (Bardi et al., 2011).  Ethanol promotes oxygen radical attack on proteins at the lipophilic side chain of amino acids (Remmer et al., 1989). Evidence for the role of oxygen free radicals in the pathogenesis of ethanol induced mucosal injury is supported by the fact that the administration of antioxidants reduces the ethanol-induced gastric injury in rat (Ligumsky et al., 1995).

1.2.2.4 Cigarette Smoking

Smoking cause’s mucosal injury by increasing the content of free oxygen radicals, platelet aggregation factor (PAF), pituitary vasopressin, gastric endothelium and pituitary vasopressin. Smoking stimulates pepsinogen secretion also by increasing chief cell numbers or with an enhancement of their secretory capacity (Walker and Taylor, 1979).

1.2.2.5 Diet

All foods are capable of stimulating gastricacid secretion through distention of the stomach, but proteins are the major stimulants. In general, amino acids, when absorbed into the circulation, stimulate acid secretion by directly stimulating parietal cells or via gastrin release in humans (Malhotra, 1978). Caffeinated beverages (e.g. tea and coffee), cola type beverages, beer and milk are potent stimulants of gastric acid secretion . A low fibre diet, high dietary consumption of salt and red/black peppers also causes peptic ulcer (Sen et al., 2009).

1.2.2.6 Psychological factor (stress ulcers)

There is evidenceconcerning the participation of reactive oxygen species(ROS) in the aetiology and physiopathology of human diseases such as neurodegenerative disorders, inflammation, viral infection autoimmune pathologies and digestive system disorders such as gastrointestinal inflammation and gastric ulcer (Reptto and Llesuy, 2002).Stress induced ulcers are due to increase in free radical generation apart from acid pepsin factors (Sairam et al., 2002). Stress causes an   increase in gastric motility, vagal over activity, mast cell degeneration, reduces gastric mucosal blood flow and increases histamine production (Sen et al., 2009). Cold restrained stress induced ulcers are the result of autodigestion of gastric mucosal barrier, accumulation of HCl and generation of free radicals (Thamotharan et al., 2010). However, oxygen-free radicals may be produced normally, as part of cellular metabolism, or as a requirement for body defence. Antioxidants act as scavengers to neutralize free radicals and have generated considerable interest in overcoming the adverse and pathological results of ROS (Ashok-Agarawa and Gupta, 2006). Various antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) control their accumulation (Fridovich, 1986).  An imbalance in the activity of these enzymes usually leads to accumulation of free radicals which has been implicated as a leading cause of lipid peroxidation and tissue damage (Bagchi et al., 1998). They are also responsible for oxidation of the bases in cellular DNA making them mutagenic, cytotoxic and crosslinking agents, which in turn cause uncontrolled expression of certain genes causing increased multiplication of cells leading to cancer (Marotta et al., 1999).  Antioxidants seemed to have protective role in gastric ulcers and carcinomas (Ito et al., 1996).  Lipid peroxidation causes loss of membrane fluidity, impaired ion transport and membrane integrity and finally loss of cellular functions. Oxidative stress also causes inactivation of prostaglandin synthetaseleading to decreased biosynthesis of prostaglandin the master molecule for gastro protectionagainst all forms of insults to the mucosa (Zhao et al., 2009).

 

Fig. 5: Pathway showing several mechanisms of free radical generation in the body.

(Source:Kunwar & Priyadarsini, 2011).

 

1.2.3 Free radicals /Reactive oxygen species

Free  radicals can be definedas molecules or molecular fragments containing one or more unpaired electrons in their atomic or molecular orbitals.This unpaired electron(s) usually gives a considerable degree of reactivity to the free radical(knight,1998).Reactive oxygen species (ROS) is a collective term used for a group of oxidants,which are either free radicals or molecular species capable of generating  free radical.Important ROS include superoxide anion,hydroxyl ion and hydrogen peroxide (Mittler,2002).Molecular oxygen (dioxygen) has a unique electronic configuration and is itself a radical.The addition of one electron to dioxygen forms the superoxide anion radical(O_2-.).Suproxide anion,arising either through metabolic processes or following addition to dietary antioxidants,the body relies on several endogenous defence mechanisms to help protect against free radical-induced cell damage.The  antioxidant enzyme ;glutathione peroxidase(GPx),catalase(CAT)and superoxide dismutase(SOD)-metabolize oxidative toxic intermediates(Niki et al., 2005) and require micronutrient cofactors such as selenium, iron, copper,zinc and manganese for optimum catalytic activity (knight,1998).

An antioxidant is a molecule that slows or prevents the oxidation of the molecules. Antioxidantsterminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often considered as reducing agents such as thiols, ascorbic acid, and polyphenols.Antioxidants are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants react with oxidants in the cell cytosol and the blood plasma, while lipid-soluble antioxidants protect cell membranes from lipid peroxidation (Sirisha et al.,2010). The body relies on several endogenuos defence mechanisms including the use of antioxidants for protection against free radical- induced cell damage.The antioxidant enzymes metabolise oxidative toxic intermediates(Niki et al.,2005) and require micronutrient cofactors such asselenium,iron,copper,zincand manganese for optimum catalytic activity(Soliman,2008).

Superoxide dismutase (SOD) is a prime antioxidant enzyme found in two forms.One complexes with zinc and copper, localized in the cytosol and the other,bound with manganese,found in the mitochondrial matrix (Soliman,2008).Both forms of this metalloenzyme catalyzes the inactivation of destructive superoxide anion by converting them to hydrogen peroxide which is then transformed to water and oxygen by the enzyme catalase(Shamal et al.,2012).

Catalase is widely distributed in all animal tissues and the highest activity is found in the red blood cells and in the liver.CAT decomposes H₂O₂ and protects the tissue from highly reactiveOH.Therefore,the reduction in the activity of these enzymes may result in an accumulation of O₂.radicals and H₂O₂.Hence,catalase has been considered an important regulator of oxidative stress(Switala and Loewen,2002 ).

Glutathione (GSH) can be used to ‘detoxify’ reactive oxygen species such as H₂O_{2 ,}  a process in which GSH is oxidised to the dimmer glutathione disulfide (GSSG) in a reaction catalysed by glutathione peroxidase (GPx)(Mukanganyama et al.,2001).Reduced glutathione(GSH) in turn is regenerated from GSSH by glutathione reductase(GR) in a reaction requiring NADPH as a cofactor.GPx reduces hydrogen peroxide to water ,along with  the oxidation of GSH and protects the erythrocyte against peroxides that are generated intracellularly or exogenously.GR plays an important role in protecting haemoglobin,red blood cell enzymes and biological cell membranes against oxidative damage by increasing the level of GSH in the process of aerobic(Mukanganyama et al.,2001).

1.3Diagnosis of ulcer

Peptic ulcer can be diagnosed either by direct visualization using an endoscope or by    using contrast radiography. The invasive methods requiring endoscopic evaluation include bacteriologic culture, susceptibility testing, histopathologic studies, molecular diagnostics, and rapid urease testing while the non-endoscopic approaches include faecal antigen detection, serologic testing, and urea breath testing.

1.3.1 Endoscopy and radiology approaches to the diagnosis of ulcer

Endoscopy is a procedure used to study the oesophagus, stomach, and duodenum using an endoscope, a long thin tube equipped with a tiny video camera. When combined with a biopsy, endoscopy is the most accurate procedure for detecting the presence of peptic ulcers, bleeding, and stomach cancer, or for confirming the presence of H. pylori (Kim et al., 2007). It is particularly helpful in identifying lesions too small to bedetected by radiographic examination (Cook et al., 1992).  Since, these endoscopic and radiographic procedures are highly expensive; attempts have been made to develop a surrogate marker for ulcer diagnosis, such as the response to an empirical trial with antisecretory agents or the evaluation of H. pylori status with serologic markers (Enaganti, 2006).

1.3.1.1Culture

Culture has the advantage of allowing for the determination of antibiotic sensitivity. Various selective and non selective media are suitable for culture, such as brain-heart infusion agar plates with other supplements and media used for Campylobacter(Cutler, 1996). Culture incubation is performed under microaerophilic conditions at 37˚C, with positive cultures usually detected after 3 to 5 days (Glupczynski, 1994; Suerbaum and Michetti, 2002). Identification of H. pylori is made on the basis of colony morphology that contains gram-negative, curved rods that test positive for urease, catalase and oxidase (Veenendaal et al., 1993).

1.3.1.2 Histological assessment

This is done using a variety of histological stains like haematoxylin and eosin. Although H. pylori is readily seen on haematoxylin and eosin (HE) stained slides of biopsy specimens, for better identification of the organism, special staining methods have been used, such as Warthin-Starry, Giemsa and Steiner silver stain, Genta stain, toluidine blue stain, carbol fuchsin stain, modified Mcmullen’s method, HpSS, methylene blue, modified Geimsa and immunohistochemical staining method (Rotimi et al.,  2000). Fluorescence in situ hybridization is a complementary technique tohistology, which is used to detect the presence of H. pylori.

1.3.1.3 Rapid urease tests

The CLO test was the first of the commercially available biopsy urease tests designed for H. pylori detection (Laine et al., 1996).  H. pylori urease hydrolyzes the urea contained in the agar gel of the test packet and leads to production of ammonia, pH rise and a colour change of the phenol red indicator (Yousfi et al., 1996). Urease also has an important role in the bacterialnitrogen cycle (Hazell and Mendz, 1997).

1.3.1.4 Polymerase chain reaction (PCR)

PCR assays, which shown to be sensitive and specific were developed for the detection of H .pylori in gastric mucosal biopsies. However, the diverse genetic organization of H. pylori may affect the sensitivity of the assay (van Doorn et al., 2000). Other investigators have amplified H. pylori DNA from human saliva, gastric juice, and faeces, although with diminished sensitivities (Gramley et al., 1999; Song et al., 1999). Quantitation of H. pylori DNA in gastric biopsy specimens was performed with the glmM gene as the target and represents an important development in molecular diagnostics for H pylori infection (He et al., 2002).

1.3.2 Non-endoscopic diagnosis of ulcer

1.3.2.1 Antibody test

Antibody tests diagnose (false negative results) and over diagnose (false positive results) H. pylori infection with some frequency. Antibody testing offers numerous advantages: (a) It is non-invasive (b) It is relatively inexpensive,  (c) It overcomes some of the limitations that identify patients with active infection such as urea breath test or stool antigen test,(d) It is fast.

1.3.2.2 Urea breath test

The urea breath test (UBT) is one of the most important non-invasive methods for detecting H. pylori infection. The diagnostic accuracy of UBT is greater than 95% (Stenstrom et al., 2008). Patients ingest 13C or 14C-labelled urea. If H. pylori are present in the stomach, urease hydrolyzes the labelled urea and releases labelled HCO₃which is transported by the bloodstream to the lungs and is exhaled as isotopically labelled carbon dioxide (Graham et al., 2001).

1.3.2.3 Stool antigen test

This test identifies H. pylori antigens in stool. This test utilizes polyclonal anti-H. pyloricapture antibody adsorbed to microwells. Diluted stool and a peroxidase conjugated polyclonal antibody are added, followed by substrate. In infected patients, enzyme-substrate binding leads to a colour change, which can be detected visually or spectrophotometrically (Gulcan et al., 2005).

1.4 Therapy for peptic ulcer

Overall treatment is aimed at relieving ulcer pain, healing the ulcer, preventing ulcer recurrence, and reducing ulcer-related complications. The goal of therapy is to eradicate the causative organism (H. pylori) andto successful heal patients with NSAID induced ulcers as rapidly as possible. Patients at high risk of developing NSAID ulcers should be switched to a COX-2 inhibitor or receive prophylactic drug co-therapy to reduce ulcer risk and ulcer related complications (Katzung, 2004; Dipiro et al., 2005).

1.4.1 Non Pharmacological treatment of peptic ulcer disease

These include;

1. Identify and instruct patients to avoid foods that cause excess hydrochloric acid secretion; this improves symptoms for some individuals.
2. Educate patients that avoidance of alcohol and caffeine improves symptoms and increases healing of a pre-existing ulcer.
3. Fibre rich diet can reduce the risk of developing an ulcer and can also speed the recovery if it already exists.
4. Flavonoid rich foods like apples, celery, cranberries, onions, garlic and tea may inhibit the growth of pylori.
5. Discontinue, reduce NSAID ingestion or switch to selective COX-2 inhibitor therapy; this often relieves symptoms in mild cases.
6. Strongly urge individuals who smoke to quit because tobacco both irritates the gut and delays healing.
7. Stress management with relaxation techniques such as yoga, or sedatives can be used to relieve psychological influences.

1.4.2 Pharmacological Treatment of peptic ulcer disease

A summary of the pharmacological Treatment of peptic ulcer disease PUD is represented below.

 

Fig. 6: Classification of various drugs used in the treatment of peptic ulcer

(Source: Gulia and Choudhary, 2011).

1.5 An overview of the immune system

The immune system consists of cells and organs that are concerned with defence of the individual, primarily against the threat of disease caused by infectious organisms.Types of immune system include adaptive and innate immune system. Innate immunity is present at birth and provides the first barrier against microorganisms there are two fundamental adaptive mechanisms: cell-mediated immunity and humoral immunity.  The adaptive immune system is antigen-specific (it recognizes and acts against particular antigens).T cells are non antibody-producing lymphocytes which are also produced in the bone marrow but sensitized in the thymus and constitute the basis of cell-mediated immunity.Although the innate and adaptive systems play different roles in defending the body, they generally act together in fighting an infection (Janeway et al., 2005).

1.5.1 Immunomodulation

Immunomodulation is a procedure which can alter the immune system of an organism by interfering with its functions.Immunomodulators have biphasic effects, some tend to stimulate immune system which is low while others inhibit host parameters which are normal or already activated (Vinothapooshan and Sundar,2011).

Immunomodulatory agents may selectively activate either cell-mediated or humoral Immunity. The primary target of the immunomodulatory compounds is believed to be the macrophages, which play a key role in the generation of an immune response. The immune system is known to be involved in the pathophysiologic mechanism of many diseases((Vinothapooshan and Sundar,2011).Whether immunomodulators enhance or suppress immune responses can depend on a number of factors such as dosage, route of administration, timing and frequency of administration.

1.5.1.1 Immunostimulation

The immune response can be modified to enhance the humoral or cell – mediated immune response against antigens.They may selectively activate either cell-mediated or humoral immunity by stimulating either TH₁ or TH₂ type cell response, respectively. Immunostimulation reinforces the immune system which primarily implies stimulation of specific and non specific system, i.e. granulocytes, macrophages, complement, certain T-lymphocytes and different effector substances.Those compounds which appear to stimulate the human immune response are being sought for the treatment of cancer, immunodeficiency diseases or for generalized immunosuppression following drug treatment, for combination therapy with antibiotics and as adjuncts for vaccines (Jong et al., 1991).

Levamisol,a well known antihelminthic has been shown to stimulate host deficiencies by modulating cell-mediated and humoral immuneresponses, enhancement of macrophage, T-cell and polymorphonuclear cell functions (Symoens and Rosentha, 1977).Levamisol does not increase the immune response above the normal level in immunologically competent host (Symoens and Rosenthal,1977).Best results from Levamisol are obtained in immunodeficient patients (Renoux, 1978, Symoenset al., 1979, Guerrero, 1980) and the effects are strictly time and dose dependent. Brunner and Muscoplat (1980) explained that though the drug is believed to potentiate immunity, its precise mechanism of action is not known. Van Eygen, et al.,1976) explained that it is apparent from clinically trials that Levamisol may be beneficial as an immunomodulator in the treatment of disease. When injected into healthy animals, Levamisol sometimes increases the number of antibody forming cells in the spleen (Renoux, 1974). However it does not markedly alter the serum antibody response of vaccination (Irwin, et al.,1976). Any effect on B- lymphocyte function is probably indirect due to stimulation of T-lymphocytes and macrophages rather than B cells themselves. The drug may enhance antigen clearance or increase the activity of suppressor lymphocytes which are important in regulation of the immune system.

1.5.1.2 Immunosuppression 

The term immunosuppression refers to impairment of the immune system resulting in decreased immune function. Immuno-suppression implies mainly to reduce resistance against infections, stress and may occur on account of environmental or chemotherapeutic factor.Clinical indicators of immunosuppression include,myelosuppression,such  pancytopenia and other blood dyscrasias, hypocellularity of immune system  tissues , decreased organ weight (Such as the thymus,spleen,lymph nodes or bone marrow), decreased serum globulin levels,increased incidence of infections, increased incidence of tumors, decreased clonal proliferation of T-cells, primarily by inhibiting IL-2 synthesis  and possibly also by  decreasing expression of IL-2 receptors , reduced induction and clonal proliferation of cytotoxic  T-cells from CD8+precursor T-cells, reduced function of the effector  T-cell that are responsible for cell-mediated  responses (example decreased delayed – type hypersensitivity),some  reduction of Tcell- dependent  B-cell responses.Examples of immunosuppressant drugs are corticosteroids, tacrolimus, azathioprine, cyclophosphamide and others (Rang and Dale’s, 2010).

Myelosuppression is defined as the bone marrow making too few blood cells to replace the blood cells that have worn out.  However, myelosuppression can lead to fatigue, excessive bleeding, or an increased vulnerability to certain infections. These three conditions are a direct result of myelosuppression, because they are a direct result of a shortage of certain blood cells and fatigue. This results from a shortage of red blood cells. It is also known as Anemia. Bleeding: This results from a shortage of platelets. It is also known as Thrombocytopenia. Infection risk: This results from a shortage of white blood cells, notably neutrophils. It is also known as Neutropenia.All of these conditions can be considered a result of myelosuppression. Bone marrow is a source of cells involved in immune activity and is the sensitive organ most affected during any immunosuppression therapy with this class of cytotoxic drugs(Cyclophosphamide). Stem cells degeneration and inability of bone marrow to regenerate new blood cells will give rise to thrombocytopenia and leucopenia (Tijani et al., 2010).

Fig.7: The structure of Cyclophosphamide

Cyclophosphamide is probably the most commonly used alkylating agent. It is inactive until metabolised in the liver by the P450 mixed function oxidases.

1.6 Humoral immunity

In the humoral immune response, B cells recognize antigens.When a B cell encounters an antigen, antigen particles will enter the B cell by endocytosis and getprocessed. Helper T cells that are able to recognize thespecific antigen will bind to the antigen-MHC protein complex on the B cell and release interleukin-2, whichstimulates the B cell to divide. In addition, free, unprocessed antigens stick to antibodies on the B cell surface. This antigen exposure triggers even more B cell proliferation. B cells divide to produce long-lived memoryB cells and plasma cells that serve as short-lived antibody factories, producing large numbers of circulating antibodies directed against those antigens. This provokes a primary response in which antibodies of the IgM class antibodies are secreted and produced first.When IgM (and to a lesser extent IgG) antibodies bind to antigens on a cell, they cause the aggregation (activation) of complement proteins (system). As we mentioned earlier, these proteins form a pore that pierces the plasma membrane of the infected cell, allowing water to enter and causing the cell to burst. The antibodies are released into the blood plasma, lymph, and other extracellular fluids (Dhasarathan, 2010).

Upon subsequent exposure, the plasma cells secrete large amounts of antibodies that are generally of the IgGclass. Because a clone of memory cells specific for that antigen develops after the primary response, the immune response to a second infection by the same pathogen is swifter and stronger. The next time the body isinvaded by the same pathogen, the immune system is ready. As a result of the first infection, there is now a large clone of lymphocytes that can recognize that pathogen .This more effective response, elicited by subsequent exposures to an antigen, is called a secondary immune response. Memory cells can survive for several decades, which iswhy people rarely contract chicken pox a second time afterthey have had it once. Although plasma cells live only a few days, theyproduce a vast number of antibodies. In fact, antibodiesconstitute about 20% by weight of the total protein inblood plasma. Production of IgG antibodies peaks afterabout three weeks. When IgG antibodiesbind to antigens on a cell, they serve as markersthat stimulate phagocytosis by macrophages. Because certaincomplement proteins attract phagocytic cells, activationof complement is generally accompanied by increasedphagocytosis (Dhasarathan,2010).

1.7 Cell mediated immunity

Upon initial exposure of rats immune system to SRBCs,  DTH requires the specific recognition of a given antigen by activated T lymphocytes, which subsequently proliferate and release cytokines. These in turn increase vascular permeability, induce vasodilatation, macrophage accumulation, and activation, promoting increased phagocytic activity and increased concentrations of lytic enzymes for more effective killing. When activated TH₁ cells encounter certain antigens,for example SRBCs, They secrete cytokines that induce a localised inflammatory reaction called delayed type hypersensitivity (DTH). DTH comprises of two phases, an initial sensitisation phase after the primary contact with SRBC antigen. During this period TH₁ cells are activated and clonally expanded by APC (antigen presenting cells) with class II MHC molecule (eg. langerhans cells and macrophages are APC involved in DTH response). A subsequent exposure to the SRBCs antigen induces the effector phase of the DTH response, where TH₁ cells secrete a variety of cytokines that recruits and activates macrophages and other non specific inflammatory mediators. The delay in the onset of the response reflects the time required for the cytokines to induce the recruitment and activation of macrophages (Dashputreet al., 2010 and Vinothapooshan G.; Sundar,K. 2011).

1.8 Anaemia

Anaemia is one of the most common haematological problems affecting people of all ages. It results from the decreased in the oxygen carrying capacity of the blood due to a reduction in the number of circulating red blood cells and also the decrease in the amount of haemoglobin present in RBCs (McGrath, 1993; Grewal,2010). Anaemia is not considered a disease in itself,rather,it reflects an abnormality in the number, structure or function of RBCs . Reduction of haemoglobin is usually accompanied by a fall in red cell count and packed cell volume(PCV) but these amount may be normal in some patients with subnormal haemoglobin levels (Hoffbrand et al.,2006). Anaemia can be a manifestation of many abnormal conditions: dietary deficiencies of iron ,vitamin B₁₂ and folic acid,bone marrow damage as a result of chemotherapy, radiation or renal disease ,malignancies, chronic infections , overactive spleen, bleeding from any organ or tract or bleeding due to cancer or traumer and hereditary disorders example sickle cell disease, thalassemia .

The incidence of anaemia is more common in underdeveloped countries because of poor nutrition and the presence of parasites that extract blood from the intestines.The severity of anaemia depends on factors such as its speed of onset whether it is chronic and the person’s overall health and nutritional status .The more rapidly anaemia develops the more likely it is to be serious(Grewal,2010).

1.8.1 Types of anaemia

1.8.1.1 Iron deficiency anaemia

Iron deficiency is the most common nutritional disorder, affecting more than one –third of the general population (Zimmermann and Hurell, 2007; Clark, 2009).Iron deficiency anaemia is the most prevalent anaemia in all age groups in the world. It results from either an inadequate absorption or an excessive loss of iron .This condition occurs most often in women, young children and older adults. Primary causes include trauma, excessive menses, bleeding from the gastrointestinal tract, pregnancy or diet that lacks iron (Zimmermann and Hurell, 2007). Deficiency caused by faulty eating habits is especially prevalent in the adolescent and elderly population.

1.8.1.2 Haemolytic anaemia

Haemolytic  anaemia results from an increase in the rate of red cell destruction .Red cell destruction usually  occurs after a mean lifespan of 120 days .Because of erythropoietic hyperplasia and anatomical extension of bone marrow , red  cell destruction may be increased several-fold before the patient becomes anaemic (Delaunay,2003).The individual will have an increase of immature RBCs(reticulocytes).The cause of haemolytic anaemia is related to defects of the cell membrane of RBC,inherited enzymes defects, certain drugs and toxins ,antibodies or physical trauma (Delaunay,2003).Haemolytic anaemia may not be noticed until the red cell lifespan is less than 30 days.Treatment of this type of anaemia relates to diagnostic and to the causative factors.

1.8.1.3 Acute haemorrhagic anaemia

Acute haemorrhagic anaemia develops after a rapid and often sudden blood loss. Causes of such blood loose  include trauma that leads to blood vessel rupture, aneurysm or artery erosion caused by a cancerous lesion or ulcer (Erber and Perry,2006) Severity and prognosis depend on the total blood volume loss.A total blood volume loss of twenty percent is considered a marked insufficiency.A loss of thirty percent will cause failure in the circulatory system, coma and shock.A loss that reaches forty percent can be fatal (Nascimento et al.,2010).Immediate treatment is volume replacement, often with administration of intravenous fluids, such as saline, albumin or plasma and tansfisions with fresh whole blood.

1.8.1.4 Chronic haemorrhagic anemia

Chronic haemorrhagic anaemia is usually the result of conditions such as peptic ulcers, bleeding haemorrhoids, excessive emesis or cancerous lesions in the gastrointestinal tract.Chronic blood loss can eventually lead to iron deficiency anaemia because available iron sources are depleted (Clark,2009).Treatment usually incudes controlling the site of bleeding and replacing lost iron through diet and supplements.

1.8.1.5 Perniciuos anaemia

Pernicous anaemia is caused by lack of a gastric susbstance called the intrinsic factor which is produced in the stomach.The body needs intrinsic factor to absorb vitamin B₁₂ from food in the small intestine (Clarke et al.,2004) .Vitamin B₁₂ is necessary for the body’s proper absorption and use of iron and the protection of nerve fibres. Perniciuos anaemia generally affects middle-aged and older adults of Northern European descent (Clarke et al., 2004). Juvenile pernicious anaemia, a rare congenital disorder in which stomach secretes abnormal intrinsic factor, generally affects children younger than 10 years.

1.8.1.6 Aplastic anaemia

Aplastic anaemia or bone marrow depression describes a condition in which the bone marrow is underdeveloped or has failed, resulting in a decreased in RBCs, WBCsand platelets (Pancytopenia) (Young, 2000).This condition may occur at any age and develop very slowly or could be rapid and very severe.Causes include excessive radiation toxicity to various drugs, tumors, insecticides,chemical and environmental toxins (Marsh,2005).It may develop as a compilation of viral infections such as hepatitis,HIV/AIDS and mononucleosis. In many cases the cause is unknown (Idiopathic aplastic anaemia).Bone marrow and stem cell transplantation is the most successful therapy.

 

 

1.9 Haematological indices

Haematological indices are the parameters that are used in the assessment of the status of blood under investigation. These parameters include red blood cells, total white blood cell, differential white blood cell, packed cell volume, erythrocyte sedimentation rate, haemoglobin count,mean cell haemoglobin concentration (MCHC),mean cell volume (MCV) and mean cell haemoglobin (MCH).

1.9.1 Red blood cells (RBC)

Red blood cells are also known as erythrocytes .The cells are flexible bioconcave disc that are involved in the transport of oxygen. The red blood cells carry haemoglobin into close contact with the tissues and for successful gaseous exchange.Red blood cells which are the 8µm in diameter , must be able to pass repeatedly through the microcirculation whose minimum diameter is 3.5µm, to maintain haemoglobin in a reduced (ferrous) state (Hoffbrand et al.,2006).

1.9.2 Packed Cell Volume (PCV)

Packed cell volume is a percentage of the known volume of whole blood occupied by packed blood cells when the blood is centrifuged at a constant speed and period of time .It is a reasonable index of the red cell population. It can equally be expressed as the fraction of the volume occupied by the erythrocytes when a sample of whole blood in a capillary or wintrobe tube is centrifuged.

1.9.3 Haemoglobin

Haemoglobin is a specialized protein that is contained in red cells of the blood .The main function of the red cells is to carry oxygen to the tissues and to return carbondioxide from the tissues to the lungs. The haemoglobin is the actual protein that performs these functions. Each red cell contains about 640 million haemoglobin molecules (Hoffbrand et al.,2006). Haemoglobin consists of four polypeptide chains, α2β2 with each containing individual haem group. Haem synthesis occurs largely in the mitochondria through a series ofbiochemical reactions.

 1.9.4 Total white blood cell (WBC)count

White blood cells also known as leukocytes are the cells of the immune system. They defend the body against pathogens, infections and foreign materials (Stock and Hoffmann, 2000). White blood cells are of two broad groups: the phagocytes and the immunocytes. The phagocytes include the three types of cell; neutrophils (polymorphs), eosinophils, basophils and monocytes. The neutrophils have a characteristic dense nucleus which consists between two and five lobes and a pale cytoplasm with an irregular outline containing many fine pink- blue (azurophilic) or grey- blue granules. The lifespan of neutrophils in the blood is only 6-10h (Hoffbrand et al.,2006).The eosinophils cells are similar to neutrophils, except that the cytoplasmic granules are coarser and more deeply red staining and there are rarely more than three nuclear lobes .The blood transit time for eosinophils is longer than that for neutrophils (Tefferi et al.,2006).They enter inflammatory exudates and have a special role in allergic responses,defence against parasitesand removal of fibrin formed during inflammation (Hoffbrand et al., 2006). The basophils are only occasionally seen in normal peripheral blood. They have many dark cytoplasmic granules which overlie the nucleus and contain heparin and histamine. The monocytes are usually larger than other peripheral blood leucocytes and possess a large central oval or indented nucleus with clumped chromatin. The cytoplasm stains blue and contains many fine vacuoles giving a ground – glass appearance.

The immunocytes include the lymphocytes, their precursor cells and plasma cells.The lymphocytes are the immunological competent cells that assist the phagocytes in defence of the body against infection and other foreign invasion.The immune response depends upon two types of lymphocytes, B and T cells, which are derived from the haemopoietic stem cell .The B cells mature in the bone marrow and circulate in the peripheral blood until they undergo recognition of antigen. T cells develop from cells that have migrated to the thymus where they differentiate into mature Tcells. Plasma cells and B lymphocytes produce a group of proteins that bind to antigen.The proteins are known as immunoglobulins and have five isotypes IgG, IgM, IgD and IgE.

1.10 Haematopoiesis

Haematopoiesis is the process of blood cells development and regulation. It involves a complex interplay between the intrinsic genetic processes of blood cells and their environment.This interplay determines whether haematopoietic stem cells (HSCs),progenitors and mature blood cells remain quiescent, proliferate,differentiate,self-renew or undergo apoptosis (Maciejewski et al.,1996) Haematopoietic agents are the substances such as iron,minerals andvitamins that are contained in foods or diets that aid in the formation of blood components.They activate the erythropoietic pathway.

1.10.1 Vitamin B₁₂

Dietary deficiency of vitamin B₁₂ occurs only in strict vegetarians,since the vitamin is found almost inclusively in animal foods.However,functional deficiency (pernicious anaemia with spinal cord degeneration)as a result of impaired absorption is relatively common,especially in elderly people with gastric gastritis (Bender 2002).The vitamin is formed by some yeasts and bacteria and there are no plant sources of this vitamin. The small amounts of vitamin B₁₂ formed by bacteria on the surface of fruits may be adequate to meet daily requirement (Bender,2002). Very small amounts of the vitamin can be absorbed by passive diffusion across the intestinal mucosa but they are not significant under normal conditions.The absorption of vitamin B₁₂is mediated by a glycoprotein, intrinsic factor.Intrinsic factor is secreted by the parietal cells of the gastric mucosa which also secretes hydrochloric acid (Bender, 2002).Gastric acid and pepsin serve to release the vitamin from protein binding and so make it available (Bender, 2002).

1.10.2 Folic acid

Folic (pteroylglutamic) acid is the parent compound of a large group of compounds,the folates(Hoffbrand et al.,2006).Humans are unable to synthesize folates and thus require preformed folate as a vitamin (Eicholzer et al., 2006).Folate functions in the transfer of one- carbon fragments in a wide variety of biosynthetic and catabolic reactions,it is therefore metabolically related to vitaminB_12. Deficiency of either vitamin has similar clinical effects and the main effects of vitamin B₁₂ deficiency are exerted by effects on  folate metabolism .Folate deficiency is thought to cause megaloblastic anaemia by inhibiting thymidylate synthesis, a rate- limiting step in DNA synthesis in which thymine monophosphate (dTMP) is synthesized as this reaction needs 5,10-methylene tetrahydrofolate (THF) polyglutamate as coenzyme (Nelson and Cox,2008,Hoffbrand etal.,2006).Every cell in the system including those of the bone marrow, require folate from  plasma as methyl THF and vitamin B₁₂ is needed in the conversion of this methyl TNF to THF which is involved in the methylation of homocysteine to methionine. Deficiency of vitamin B₁₂ prevents the demethylation of methyl THF thus depriving cells of THF and so of folate polyglutamate coenzymes. The resultant is in the reduction of the four nucleotides required for the synthesis of DNA.

Although folate is widely distributed in foods,dietary deficiency is not uncommon and a number of commonly used drugs can cause folate depletions(Bender,2002).Natural folates found in foods are all conjugated to a polyglutamyl chain containing different numbers of glutamic acids depending on the type of food (Scott and Weir,1994).

1.10.3 Vitamin C (Ascorbic Acid)

Scurvy is a disease that is associated with vitamin C or ascorbic acid deficiency. Vitamin C is required among other things for the hydroxylation of proline and lysine in collagen .Scurvy is a deficiency disease characterized by general degeneration of connective tissue.Manifestation of advanced scurvy include numerous small haemorrages caused by fragile blood vessels, toothloss, poor wound healing and the reopening of old wounds,bone pain and degeneration and eventually heart failure (Nelson and Cox,2008).  It has been established that vegetables and fruits are the major sources of antioxidants, vitamins and minerals (Aguwa and Ukwe, 1997). The presence of these vitaminsin the leave extract could suggest the possible role in curbing the incidence of oxidative stress in both humans and animals. Anaemia is frequently associated with scurvy and may be either macrocytic, indicative of folate deficiency or hypochromic,indicative of iron deficiency (Bender,2008). Iron deficiency in scurvy could be due to the reduced absorption of inorganic iron and impaired mobilization of tissue iron reserves. At the same time, the haemorrages of advanced scurvy will cause a significant loss of blood.

1.10.4 Iron

Iron is essential to all cells. The most obvious function of iron is in the haem of haemoglobin where it plays a role as the oxygen-carrying component in red blood cells and myoglobin in muscles (Provan,1999, Beard, 2011).Haem is also important as the coenzyme for oxidation and reduction reactions in a variety of enzymes including the cytochromes. A number of enzymes also contain non haem iron (i.e iron bound to the enzymes other than in haem) which is essential to their function (Bender, 2008).Iron is also involved in energy metabolism, gene regulation, cell growth and differentiation,neurotransmitter synthesis and protein synthesis (Beard,2011).

Deficiency of iron leads to reduced synthesis of haemoglobin and hence a lower amount of haemoglobin in red blood cells. Iron deficiency anaemia is a major problem world wide, especially among women (Looker et al., 1997) .Iron in foods occurs in two forms, haem in meat and meat products and inorganic iron salts in plant foods. The absorption of haem iron is better than that of inorganic iron salts, only about ten percent of the inorganic iron of the diet is absorbed although this is increased by vitamin C (Bender,2008). Iron is absorbed from the gastrointestinal tract and transported in the blood bound to transferring.Iron absorbed as haem is broken down to release iron. Inorganic iron absorption is favoured by factors such as acid and reducing agents that keep iron in the gut lumen in the Fe²⁺ rather than the Fe³⁺ state. The protein DMT-1 (divalent metal transporter 1) is involved in the transfer of iron from the lumen of the gut across the enterocyte microvilli (Hoffbrand et al., 2006) .Excess iron is stored primarily in the liver,bone marrow and spleen as ferritin (Wu et al.,2002).

1.10.5 Erythropoietin (EPO)

Erythropoietin(EPO) is a growth factor ,the primary regulator of erythropiesis,it is a glycoprotein hormone produced initially in  the foetal liver and thereafter in the kidneys (Lacombe and Mayeux,1999,Fisher.2003).It is  heavily glocosylated with approximately forty percent of the molecule composed of sialic acid .These carbohydrate moieties are important in the biologic activity and stability of EPO ,as  desialation reduces the half – life of EPO in the circulation.EPO is a hydrophobic molecule and requires intact disulfide bonds for activity (Ridley et al., 1994). Nonetheless, the structure of the active site remains to be fully characterized. Hypoxia stimulates production of EPO and the elevation of serum EPO levels in response to hypoxia is a transient process, such as increased EPO concntration eventually will return to normal even if hypoxia persists.Erythropoietin increases the number of developing erythroid precursors and accelerates release of reticulocytes from the bone marrow, but does not alter cell-cycle length or the number of meiosis involved in erythroid differentiation in stimulating such activity. EPO acts synergistically with other cytokines to promote the proliferation ,differentiation and survival of progenitor cells of the erythroid lineage and thus  boosts the production of red blood cells (Reissman and Udupa,1982,Diamante- Kandarakis ,2005 ).There is additional evidence that EPO also increases the pool of cells capable of erythroid differentiation (Reissman and Udupa,1982). Haematopoiesis begins with primitive pluripotent stem cells differentiating into multipotent prodenitor cells while EPO is the important hormone that influences erthroid differentiation (Gregory et al., 1973, Magli et al., 1982).It does not seem to interact with pluripotent or multipotent haematopoietic progenitor cells  that is  EPO –responsive cells do not seem to arise directly from pluripotent stem cells and are not capable of self renewal.

1.11 Phytochemicals

Phytochemicals are a heterogenuos group of chemicals compounds with numerous biologically active plant compounds that have potential disease inhibiting capabilities (Akinmoladun et al., 2012). It deals with variety of organic substances that accumulates in plants. They comprise a number of substances such as phenolics (flavonoids, anthocyanins, catechins, etc, carotenoids (carotene, lutein, lycopene, etc), sterols (campesterlos, sitosterol, stigmasterol and alkaloids (caffeine, theobromine, theophylline, etc). Phytochemicals are found in fruits, vegetables, seeds and herbs.These complex materials are refered to as secondary metabolites (Akinmoladun et al., 2012).

1.11.1 Phytochemical constituents in plants

Phytochemicals are chemical compounds formed during the plant normal metabolic processes.These chemicals are often referred to as ‘secondary metabolites’ of which there are several classes including alkaloids, flavonoids, coumarins ,glycosides ,gums, polysaccharides, phenols, tannins, terpenes and terpeniods (Harborne,1998; Okwu,2004).Phytochemicals are present in a variety of plants utilized as important components of both human and animal diets.These include fruit,seeds,herbs and vegetables (Okwu,2005). Phytochemicals are naturally occurring and believed to be effective in combating or preventing disease due to their antioxidant effect (Halliwell and Guttteridge, 1992, Farombi et al.,1998).The medicinal values of these plants lie in their component phytochemicals which produce the definite physiological actions on the 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-carsinogenic,immunostimulators and some possess other beneficial properties, some prevent oxidation by free radicals and therefore known as chemopreventers. Antioxidant activity is one of the predominant mechanisms of their protective action and is due to their capacity to scavenge free radicals. Among the most investigated chemopreventers are some vitamins, plants polyphenols and pigments such as carotenoids, chlorophylls, flavonoids and betalains (Dandjesso et al.,2012)

1.11.1.1 Alkaloids

Alkaloids are one of the most diverse groups of secondary metabolites found in living organisms and have an array of structural types, biosynthetic pathways and pharmacological activities (Roberts and Wink, 1998).Although alkaloids have been traditionally isolated from plants, an increasing number is found in animals, insect marine invertebrates and microorganisms. Alkaloids generally exert pharmacological activity particulary in mammals such as humans  Even today many of our most commonly used drugs are alkaloids from natural sources and new alkaloidal drugs are still being developed for clinical use (Roberts and Winks ,1998).Most alkaloids with biological activity in humans affect the nervous system, particulary the action of neural transmitters example acetylcholine,adrenaline noradrenaline,gamma-aminobutyric acid (GABA),dopamine and serotonin (Schmeller and Wink,1998). Most alkaloids are physiologically active compounds having a variety of toxic effects on animals (Roberts and Wink, 1998). Toxic alkaloids are frequently found as part of conspicuous, often violent, insect defence systems and these may be synthesized by insects or acquired as part of their diet.However, plants sequester alkaloids for use as a passive defence mechanism by acting as feeding deterrents for predating insects.

1.11.1.2 Flavonoids

Flavonoids are also referred to as bioflavonoids.They are antioxidants that can contain polyphenols.They are organic compounds that have no direct involvememt with the growth or development of plants.Flavonoids are plant nutrients when consumed in fruits and vegetables pose no toxic effect on humans and are also beneficial to the human body.Flavonoids are widely distributed throughout plants and they give flowers and fruits of many plants their vibrant colours.They also play an important role in protecting the plants from microbes and insect attacks.More importantly,the  consumption of  foods containing flavonoids has been linked to numerous health benefits.Though research shows flavonoids alone provide minimal antioxidant benefit due to their absorption by the body,it has been identified that they biologically trigger the production of natural enzymes that fight pathogens and diseases.Flavonoids can be nutritionally helpful by triggering enzymes that reduce the risk of certain cancers,heart diseases and age – related degenerative diseases.Some research also indicates flavonoids may help prevent tooth decay and reduce the occurrence of common ailments such as the flu. Chinese and Japanese scientists have known the anti-ulcer, detoxifying and antioxidant activities of licorice flavonoids for at least two decades. The anti-ulcer effects have been confirmed in humans and products containing licorice flavonoids are now being marketed in China and Japan for treating ulcers.  Their effects are reported to be at least equal to those of Ranitidine, but without the toxic side effects of the latter.

1.11.1.3 Tannins

Tannins are polymerized phenols with defensive properties. Their name comes from their use in tanning rawhides to produce leather.In tannin, collagen proteins are bound together with phenolic groupsto increase the hides resistance to water, microbes and heat (Heldt and Heldt, 2005).Two categories of tannin that are of importance are the condensed and hydrolysable tannins .The polymerization of flavonoid molecules produces condensed tannins, which are commonly found in woody plants. Hydrolyzable tannins are also polymers but they  are more heterogeneous mixture of phenolic acids (especially  gallic acids) and simple sugars.Thuogh widely distributed, their highest concentration is in the bark and galls of oaks(Heldt and Heldt,2005). Tannins are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins and various other organic compounds including amino acids and alkaloids (Petridis,2010).The astringency from tannins is what causes the dry and puker feeling in the mouth following the consumption unripe Fruit or red wine (Serafini et al.,1994). Many human physiological activities such as stimulation of phagocytic cells,host- mediated tumour activity and a wide range of antiinfective action ,have been assigned to tannins (Haslam,1996).One of their biological actions is to complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic interactions as well as by covalent bond formation (Haslam,1996,Stern et al.,1996).Thus,their mode of antimicrobial action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope, transport proteins etc.

1.11.1.4 Steroids

Steroids are triterpenes which are based on the cyclopentane hydrophenanthrene ring system (Harborne, 1998).Sterols were at one time considered to be animal substances  (Similar to sex hormones,bile acids,etc.) but in recent years ,an  increasing number of such compounds have been detected in plants tissues.Sterols have essential functions in all eukaryotes.For example,free sterols are integral components of the membrane lipid bilayer where they play an important role in the regulation of membrane fluidity and permeability (Corey et al.,1993).While cholesterol is the major sterol in animals ,a mixture of various sterols is present in higher plants with three known types occurring in higher plants: sitosterol (formerly known as B- sitosterol),stigmasterol and campesterol (Harborne,1998).These common sterols occur both free and as simple glucosides. Certain sterols are confined to lower plants, one of which is regosterol, found in yeast and many fungi.Others occur mainly in lower plants but also appear occasionally in higher plants example fucosterol,the main steroid of many brown algae and also detected in coconut (Harborne,1998).

1.11.1.5 Terpenoids

Terpenoids are secondary plant metabolites.Terpenoids form a group of compounds the majority which occur in the plant kingdom,a few terpenoids have been obtained from other sources (Singh,2007).Most plant hormones are derivatives of terpenoids which include cytokinins,gibberellins and abscisic acid (Harrawijn et al.,2001).The steroid hormones of mammals are terpenoids with an advanced but not very complex structure. Many terpenoids are produced via the mevalonic acid pathway others are biosynthesized via a recently discovered pathway,a mevalonate independent route.The simpler mono-and sesqui-terpenoids are the chief constituents of the essential oils, these are the volatile oils obtained from the sap and tissues of certain plants and trees (Singh,2007).The essential oils have been used in perfumery  from the earliest times .The di- and tri- terpenoids which are not  steam volatile  are obtained from plant and tree gums and resins. The tetraterpenoids form a group of compounds known as the caroteniods (Singh, 2007).

 1.11.1.6 Saponins

Saponins are glycosides of triterpenes and steroids which are characterized by bitter or astringent taste , forming  properties (Okigbo et al., 2009) haemolytic effect on red blood cells and  cholesterol binding properties (Okwu,2005).Saponins increase the permeability of intestinal mucosa cells, inhibit active nutrient transport and facilitate the uptake of substances to which the gut would normally be impermeable (Gee et al.,1997) .It has also been shown  to possess beneficial effects such as cholesterol  lowering properties and exhibits structure dependent biological activity (Harbone,1998). Saponins, being both fat and water soluble, have surfactant and detergent activity. Thus they would be expected to influence emulsification of fat-soluble substances in the gut including the formation of mixed micelles containing bile salts, fatty acids and fat –soluble vitamin. Saponins are involved in ulcer protection and certain antimicrobial activity.

1.12 Medicinal plants

Man has been using herbs and plant products for combating diseases since time immemorial. The traditional system of medicine has been such that larger percentage of the populations in Africa and Asia depend on indigenous system for relief of symptoms.With such a huge section of an ever-increasing population relying on herbal remedies, it is imperative that plant products which have been in use from ages have scientific support for their efficacy (Deepanjana et al., 2011).Generally, drugs used in medicine are either obtained from nature or are of synthetic origin. Ayurvedic medicinal plants have been shown as the basis for production of new drugs and have great promises (Mukherjee,2002).The WHO has shown great interest in plant derived medicines which have been described in the folklore medicines of various countries (Mukherjee,2002).

Despite the breakthroughs in disease management and medicine, disease causing microorganisms continually undergo genetic variations which permit them to survive and resist treatment especially with antibiotics.Hence, even in this modern age the search for newer sources of antimicrobial agents in disease management continues to be important (Adebayo-Tayo and Odeniyi 2012)

 

1.12.1 Ficus capensis

1.12.1.1 Botanical outline of Ficus capensis

Ficus plants belong to the mulberry family, Moraceae. Ficus capensis, also known as the cape fig and is a native of tropical Africa and the Cape Islands. The plant is a deciduous tree with spreading roots and branches and broad green leaves. The name “fig” is derived from the hollow, pear-shaped inflorescence called “Syconia”. F. capensis produces fleshy fruits all year round in a single or branched raceme along the trunk and the main braches. These fruits, though inedible to humans are eaten by many animals. Keay (1989) described F.capensis as having ovate to elongated elliptic leaves, about 7.5 – 15 cm long and 5 -10 cm wide. The leaves have acute or blunt apex with a slightly cordate, sometimes rounded, occasionally cuneate or slightly unequal asymmetrical base. They are some what leathery, dark green with glabrous surfaces with no epidermal hairs. The margin could be dentate, wavy or at times, entire. The fruits are usually densely clustered along short branched shoots on the main stem and older branches. Solitary or paired figs may be found among the leaves. The individual figs have short stalks 2.5 – 3 cm long.Ficus capensis is known by Igbos as Akokoro Yorubas as Opoto, Hausas as Uwaraya ,Fulanis asRimabichei,Edos as Obada(Otitoju et al.,2014)

1.12.1.2 Uses of Ficus capensis

Many of the world’s population are versed in the use of the available plants and herbs in their environment in the treatment of different diseases . In Nigeria, andother parts of the world, the plant along with many others in this family is important in the traditional treatment of many diseases and ailments. The plant extracts have been reported in the treatment of diarrhea, dysentery, sexually transmitted disease causing microorganisms, chest ailments, tuberculosis, leprosy, convulsions, pain, in anaemia and wound among many others (Sirisha et al ., 2010).

The relevance of this plant in traditional medicine is as a result of the secondary metabolites such as phytates, phenols, saponins, tannins, alkaloids, terpenoids and flavonoids which they have been screened to contain. Also referred to as phytochemicals, they are reported to possess inhibitory activities against the growth and disease inducing activities of some pathogenic microorganisms (Solomon-Wisdom et al., 2011). Ficus species are rich source of polyphenolic compounds, flavanoids which are responsible for strong antioxidant properties that help in prevention and therapy of various oxidative stress related diseases such as neurodegenerative and hepatic diseases (Sirisha et al.,2010).

Ficus capensis (Moraceae) leaves have been reported in literatures topossess anti-diarrhea properties and have been used as such by herbal practitioners in some parts ofBenue State, Nigeria (Ayinde andOwolabi, 2009). Gill (2007) reported theuse of the plant leaves in treating dysentery, oedema, epilepsy and rickets in infants among some tribes in Edo-Delta areas.There are reports on the antimalarial, antibacterial and anti- ulcer activities of the leaves . The leaves of Ficus capensis (Akokoro) were reported to be used in preparing pottages of yam and cocoyam in Ngwo village, Udi local government area of Enugu state(Otitoju et al.,2014).. Plate 1: Ficus capensis

1.12.1.3Taxonomy of Ficus capensis

Kingdom:                    Plantae

Phylum:                       Magnoliophyta

Class:                           Magnoliopsida

Order:                          Rosales

Family:                        Moraceae

Genus:                         Ficus

Species:                       Ficus capensis

(Source:Sirisha et al.,2010)

1.13Aim and objectives of study

1.13.1 Aim of study

This study is aimed at validating the traditional use of the leaves of Ficus capensis in folk medicine as an anti- ulcer and immunostimulatory agent.

1.13.2 Specific objectives of the study

The specific objectives of this research include:

1. To determine qualitative and quantitative phytochemical constituents of the aqueous extract of Ficus capensis leaves.
2. To determine the lethal dose (acute toxicity) of the aqueous leaves extract which will help to detect the actual effective and therapeutic dose safe for use.
3. To determine the effect of extract on the concentrations and activities of some ulcer biochemical markers (MDA, Catalase, SOD and glutathione). This will help to explain the biochemistry behind the antioxidant activity of theaqueous extract of Ficus capensis leaves.

4. To determine the effects of the extract ondelayed type hypersensitivity(DTH) reactions, Humoral antibody synthesis, Cyclophosphamine- induced myelosuppression using haematological profile such as WBC,Hb,PCV and RBC

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