ANTI-ULCER ACTIVITY OF AQUEOUS TUBER EXTRACT OF Cyperus esculentus ON DICLOFENAC SODIUM AND HISTAMINE INDUCED GASTRIC LESIONS IN WISTAR ALBINO RATS

CHAPTER ONE

INTRODUCTION

Acid facilitates the digestion of protein and absorption of iron, calcium, and vitamin B-12 as well as preventsbacterial overgrowth and enteric infection (Schubert and Shamburek, 1990). However,when levels of acid (and pepsin) overwhelm mucosaldefense mechanisms ulcers occur.Peptic ulcer disease occurs mainly due to consumption of NSAIDS, infection by H. pylori, stress or due to pathological condition such as Zollinger-Ellison Syndrome (Mohammed et al., 2008).It has been shown that long term use ofulcer drugs may be associated with ineffectiveness ofdifferent drug regimens and even resistance to drugs isemerging (Al-Mofleh et al., 2007). Thus, there is anurgent need to identify more effective and safe anti-ulceragents. In the scientific literature, a large number of medicinal plants with gastric anti-ulcer potential have been reported(Ketuly et al., 2011). Plant extracts are some of the most attractive sources of new drugs and have been shown to produce promising results for the treatment of gastric ulcer (Schmeda-Hirschmann and Yesilada, 2005). One such medicinal plant is Cyperus esculentus (Borges et al., 2008).

 

1.1      Cyperus esculentus

Cyperus esculentus (Tigernut) is an underutilized plant of the family Cyperaceae, which produces tubers from the base that are somewhat spherical (Cortes et al., 2005). The plant is not really a nut but a tuber first discovered some 4000 years ago (Lowe and Whitewell, 2000). It has other names like yellow nutsedge, chufa, flatsedge, rush nut, water grass, earth almond, northern nut grass and nut grass (Shilenko etal., 1979). Cyperus esculentus is known in Nigeria as aya in Hausa, ofio in Yoruba and akihausa in Igbo. Cyperus esculentus grows mainly in the middle belt and northern regions of Nigeria (Okafor et al., 2003), where three varieties (black, brown and yellow) are cultivated (Umerie et al., 1997). Among these, onlytwo varieties, yellow and brown are readily available in the market. The yellow variety is preferred to all other varieties because of its inherent properties like its bigger size, attractive colour and fleshier body (Belewu and Abodurin, 2006). Cyperus esculentus can be eaten raw, roasted, dried, baked or be made into a refreshing beverage called kuunu (Oladele and Aina, 2007).

 

 

1.1.1      Biochemical and Pharmacological benefits of Cyperus esculentus

Cyperus esculentus was reported as healthy food and helps in preventing heart failures, thrombosis and poor blood circulation. It helps in preventing cancer, due to high content of soluble glucose. It was also found to assist in reducing the risk of colon cancer (Adejuyitan et al., 2009).  Cyperus esculentus is rich in energy content (starch, fat, sugars and protein), mineral (phosphorus, potassium) and vitamins E and C (Belewu and Belewu, 2007). Cyperus esculentus is suitable for diabetic persons and also helps in weight loss (Borges et al., 2008). Its tubers are said to be aphrodisiac, carninative, diuretic, emmanagogue and stimulant (Chevallier, 1996). In addition, it has been demonstrated to contain very high essential amino acids than those proposed in the protein standard by FAO/WHO/UNU (1985) for satisfying adult needs (Bosch et al., 2005). Cyperus esculentus milk has been found to be good for artherosclerosis. It contains arginine, a precursor of nitric oxide that helps the vein to relax and dilate. Its milk without sugar can be drunk by diabetics for its content in carbohydrates which is a base for sucrose and starch (without glucose) and due to its content of arginine which liberates the hormone that produces insulin (Adejuyitan, 2010).

 

Table 1: Proximate composition of Cyperus esculentus tuber

Parameter                       Dry matter (%)

Moisture                         (%wet wt) 5.77 Crude protein                                  7.00 Ether extract                                    25.70 Total ash                                          1.86 Crude fibre                                       5.50 Total carbohydrate                          65.50 Caloric value (kcal)                         524.6

Source: Oderinde and Tairu, 1988.

 

	1.1.2 Taxonomic Hierarchy of Cyperus esculentus
	KingdomPlantae  – plantes, Planta, Vegetal, plants Subkingdom                       Viridaeplantae  – green plants Infrakingdom                   Streptophyta  – land plants Division                      Tracheophyta  – vascular plants, tracheophytes Subdivision              Spermatophytina  – spermatophytes, seed plants Infradivision          Angiospermae  – flowering plants, angiosperms
Class Superorder   Order Family   Genus Species		Magnoliopsida Lilianae  – monocots, monocotyledons, monocotylédones Poales Cyperaceae  – sedges, foins coupants, laîches, rouches, rouchettes Cyperus L. – nutgrass, flatsedge Cyperus esculentus L. – chufa flatsedge, chufa, yellow nutgrass, yellow nutsedge

Source:  (Cyperaceae of North America, 2010).

 

1. esculentus plant C. esculentus seed

 

Figure1.Cyperus esculentus plant and the seed.

Source: (Cyperaceaeof North America,2010)

 

 

 

 

 

1.1.3         Phytochemistry of Cyperus esculentus

As reported by Ekeanyanwu et al. (2010), the presence of alkaloids, cyanogenic glycosides, resins, tannins, sterols and saponins were observed in the raw tuber. However, only alkaloids, sterols and resins were observed in the roasted tuber. Analysis of the antinutrient composition yielded oxalates (0.25±0.65 g/100 g), phytate (1.97±0.81 mg/100 g), saponins (0.88±0.02/100 g), tannins (9.50±0.46 mg/100 g) and cyanogenic glycosides (1.80±0.69 mg/100 g). Roasting numerically decreased the levels of the anti-nutritive factors.

 

1.2       Definition of Ulcer

An inflammatory, usually suppurating, lesion on skin or internal mucous surface that results in necrosis is referred to as ulcer (Nwodo, 2012). It could be also defined as disruption of the mucosal integrity of the stomach and/or duodenum leading to a local defect or excavation due to active inflammation. The term ‘peptic ulcer’ also known as peptic ulcer disease (PUD) refers to those ulcers that occur in either the stomach or the first part of the small intestine that leads out of the stomach, called the duodenum (Rang et al., 2003). Peptic ulcers are open craters or sores that develop in the inner lining (mucosa) of the stomach or the duodenum (the first section of the small intestine).Pathophysiology of ulcer is due to an imbalance between aggressive factors (acid, pepsin, h. pylori and non-steroidal anti-inflammatory agents) and local mucosal defensive factors (mucus, bicarbonate, blood flow and prostaglandins). Integrity of gastroduodenal mucosa is maintained through a homeostatic balance between these aggressive and defensive factors (Raskin et al., 2006).Three main factors play a role in the epithelial defense system: a hydrophobic surface barrier created by mucous layer, a pH gradient above the epithelium due to bicarbonate secretion, and epithelial cell barrier itself.

 

1.2.1     Epidemiology of Peptic Ulcer

Approximately 500,000 persons develop peptic ulcer disease in the United States each year. (Kurate et al., 1992) In 70% of patients it occurs between the ages of 25 and 64 years (Sonnenberg and Everhart, 1996). Approximately 4,500 people in the UK and 15,000 people in the US die in the complications of PUD each year. Its lifetime prevalence rate of 13% in male and 11% in female with an average annual incidence of 1.7 % per 1000 cases has been recorded (Kurate et al., 1992). However, the incidence of peptic ulcers is declining, possibly as a result of the increasing use of proton pump inhibitors and decreasing rates of Helicobacter pylori infection (Kang et al., 2002).

 

1.2.2      Symptoms of stomach ulcer

The most common ulcer symptom is gnawing or burning pain in the epigastrium. This pain typically occurs when the stomach is empty, between meals and in the early morning hours, but it can also occur at other times. It may last from minutes to hours and may be relieved by eating or by taking antacids.Less common ulcer symptoms include nausea, vomiting, and loss of appetite. Bleeding can also occur; prolonged bleeding may cause anemia leading to weakness and fatigue. If bleeding is heavy, hematemesis, hematochezia, or melena may occur.

 

1.2.3    Mechanism of acid secretion

The principal stimulants of acid secretion are histamine, released from ECL cells (paracrine); gastrin, released from G cells (hormonal); and acetylcholine released from postganglionic enteric neurons (neurocrine) (Figure 2). These agents interact with receptors coupled to 2 major signal transduction pathways: adenylate cyclase in the case of histamine and intracellular calcium in the case of gastrin and Ach (Figure 2). Histamine, released from enterochromaffin-like (ECL) cells, binds to H2 receptors that activate adenylate cyclase (AC) and generate cAMP. Gastrin, released from G cells, binds to CCK2 receptors that activate phospholipase C to induce release of cytosolic calcium (Ca²⁺). Gastrin stimulates the parietal cell directly and, more importantly, indirectly by releasing histamine from ECL cells. Ach, released from intramural neurons, bind to M3 receptors that are coupled to an increase in intracellular calcium. The intracellular cAMP- and calcium-dependent signaling systems activate downstream protein kinases ultimately leading to fusion and activation of H⁺K⁺-ATPase, the proton pump. The main inhibitor of acid secretion is somatostatin, released from oxyntic and pyloric D cells (paracrine). Each of these agents acts directly on the parietal cell as well as indirectly by modulating the secretion of neuroendocrine cells.

 

 

Figure 2:Model illustrating parietal cell receptors and transduction pathways.

Source: (Mitchell and David, 2008).

 

1.2.4         Pathogenesis of Ulcer

“No gastric acid, no peptic ulcer” is a misconception. Injury to gastric and duodenal mucosa develops when deleterious effects of gastric acid overwhelm the defensive properties of the mucosa. (Mitchell and David, 2008) Inhibition of endogenous prostaglandin synthesis leads to a decrease in epithelial mucus, bicarbonate secretion, mucosal blood flow, epithelial proliferation, and mucosal resistance to injury. (Figure 3)

Figure 3. Pathogenesis of peptic ulcer disease.

Source: Kumar et al. (2007).

 

The factors that produce ulceration by aggravating gastric acid and pepsin secretion are categorized into two:

1.2.4.1   Endogenous factors: These include different visceral neurotransmitters / hormones

(histamine, gastrin and cholecytokinin, acetylcholine), H⁺K⁺-ATPase,second messengers (Ca²⁺), genetic factors and zollinger -ellison syndrome.

• Histamine

Histamine, produced in ECL cells by decarboxylation of L-histidine by histidine decarboxylase (HDC), stimulates the parietal cell directly by binding to H2 receptors coupled to activation of adenylate cyclase and generation of adenosine 3’, 5’-cyclic monophosphate (cAMP) (Soll and Wollin, 1979). Histamine also stimulates acid secretion indirectly by binding to H3 receptors coupled to inhibition of somatostatin and thus stimulation of histamine and acid secretion (Vuyyuru et al., 1995). Histamine does not only enhance gastric acid secretion, but it also causes disturbances of the gastric mucosa, microcirculation, abnormal motility, and reduction in mucus production. The mechanism by which histamine induces gastric ulcers is through its potent acid stimulating and vasodilating capability, which leads to increased vascular permeability(Cho and Pfeiffer, 1981).  Considering the gastric parietal cells, histamine interacts with H₂ receptors and initiates a second messenger response which proceeds by increasing adenylyl cyclase activity which results in an increase in the second messenger, cyclic AMP.The cyclic AMP causes an increase in intracellular calcium levels.  This releasing characteristic of calcium applies broadly in physiology.

 

• Gastrin and cholecytokinin

Gastrin is synthesized as a large precursor molecule of 101 amino acids. Gastrin and cholecystokinin (CCK) possess an identical carboxyl-terminal pentapeptide sequence (-Gly-Trp- Met-Asp-Phe-NH2). Two main classes of gastrin/CCK receptors have been characterized: CCK1 (formerly CCK-A) and CCK2 (formerly CCKB or CCKB/gastrin). CCK1 receptors are specific for CCK, whereas CCK2 receptors recognize both CCK and gastrin with high affinity. CCK2 receptors have been identified on human parietal and ECL cells where they are coupled to activation of phospholipase C and release of intracellular calcium. (Schmitz et al., 2001) It seems that intracellular concentrations of cAMP must first be above a threshold before gastrin can directly stimulate the parietal cell. (Geibel et al., 1995)

Regulation of Gastrin

At least 2 negative feedback pathways, mediated via release of somatostatin, regulate gastrin secretion. The first is activated by luminal acidity. In rats, it involves sensory CGRP neurons. Low intragastric pH (high intragastric acidity) activates CGRP neurons that, via an axon reflex, stimulate somatostatin and thus inhibit gastrin secretion (Manela et al., 1995). Conversely, when intragastric pH rises (low intragastric acidity), somatostatin secretion is inhibited, and patients develop hypergastrinemia. The second negative feedback pathway involves a paracrine pathway whereby gastrin directly stimulates somatostatin and thus attenuates its own secretion. (Schubert et al., 1991)

 

• Acetylcholine (Ach)

Muscarinic receptors on parietal cells are of the M3 subtype. Like CCK2 receptors, M3 receptors are coupled to activation of phospholipase C with generation of inositol triphosphate and release of intracellular calcium (Figure 2). (Kajimura et al., 1992) Alcoholic beverages produced by fermentation stimulate gastric acid secretion, and the effect may be mediated via activation of M3 receptors (Yamaji et al., 2007).Ach also stimulates acid secretion indirectly by activating M2 and M4 receptors on D cells coupled to inhibition of somatostatin secretion, thus removing the tonic restraint exerted by this peptide on gastrin, ECL, and parietal cells.

 

• H⁺K⁺-ATPase

Parietal cell secretion is increased by activation of intracellular cAMP and calcium-dependent signaling pathways that activate downstream protein kinases, ultimately leading to fusion and activation of H⁺K⁺-ATPase, the proton pump (Figure 2). This enzyme, which consists of 2 subunits, catalyzes the electroneutral exchange of luminal K⁺ for cytoplasmic H⁺. Upon stimulation, these vesicles fuse with the apical plasma membrane, resulting in extensive infoldings. Upon cessation of secretion, the H⁺K⁺-ATPase is retrieved from the apical membrane, and the tubulovesicular compartment is reestablished. The precise mechanisms regulating trafficking are not known, but data suggest that it involves actin-based microfilaments, small GTPases, docking/fusion proteins, cytoskeletal linkers, and clathrin (Karvar et al., 2002).

 

• Calcium ion(Ca²⁺) as a second messenger

Calcium ions play an important role in many exocrine and endocrine tissues. There is evidence that both gastric acid secretion and gastrin release are calcium dependent processes (Figure 2).

The administration of calcium both orally or intravenously, stimulates acid secretion and increases circulating concentration of gastrin (Petersen and Maruyama, 1984). The effect of histamine is mediated by increasing adenylate cyclase activity, whereas the effects of the acetylcholine and gastrin seem to involve an increase in cytosolic free calcium (Zhou et al., 1997).

 

 

 

• Gastrinoma /Zollinger-Ellison Syndrome (ZES)

ZES is a clinical syndrome due to the ectopic secretion of gastrin by a neuroendocrine tumor (gastrinoma), located primarily in the duodenum (60%-80%) or pancreas (10%-40%), resulting in gastric acid hypersecretion. If left untreated results in refractory peptic ulcer disease, severe gastroesophageal reflux disease, diarrhea and finally death, primarily due to the complications of the refractory peptic ulcer disease (Ellison and Johnson, 2009).

 

• Genetic Factor

Approximately 20–50% of duodenal ulcer patients report a positive family history; gastric ulcer patients also report clusters of family members who are likewise affected.

 

• Blood group O

The results of the work done by Roberts et al., showed that the incidence of duodenal ulcer was 1.38 times in people belonging to blood group O as compared to those having other blood group (Roberts, 1957). More rigorous investigation into the relation led to the observation that some people secrete ABH antigens in their body fluids. Further research proposed that people belonging to blood group O might be producing a greater amount of hydrochloric acid which could be a cause of duodenal ulcer in future (Clarke, 1959). Later, it was found that in people with blood group O, there was increased binding of Helicobacter pylori to the epithelial cells. It was concluded that denser colonization of epithelial cells and increased inflammatory reactions as a result of bacterial binding in group O people might be one reason that this population is more prone (Alkout et al., 2000). A study looking into the mechanism of this association showed that serum-pepsinogen level was found to be greater for individuals with blood type O than blood type A. It is believed that the variation in quantity of serum-pepsinogen is in relation to the size of gastric secretory-cell mass. It is hypothesized that blood group culminates the development of secretory cell mass, reinforcing that gastric peptic cell mass is larger in group O. This might be one of the reasons why blood group O is more susceptible to ulcers (Hanley, 1964).

1.2.4.2 Exogenous factors: These include bacterial infection (H. pylori), NSAIDs, alcohol, psychogenic factors and dietary habits.

 

• Helicobacter pylori( pylori)

1. pyloriis a spiral, gram- negative, microaerophilic, rod-shaped bacterium with multiple flagella. H. pylori causes more than 90% of duodenal ulcers and up to 80% of gastric ulcers. It is found in the gastric mucous layer or adherent to the epithelial lining of the stomach. It produces ammonia which maintains a neutral microenvironment around the bacteria. It is highly motile and produces the enzyme urease to alter the surrounding pH so as to protect itself from gastric acid. The decrease in acid secretion is thought to facilitate survival of the organism and colonization of the stomach (Meyer-Rosberg et al., 1996). The mechanism whereby H. pylori inhibits acid secretion is multifactorial and includes direct inhibition of the parietal cell (and perhaps ECL cell) by a constituent of the bug (eg, vacuolating cytotoxin, lipopolysaccharide, or acid-inhibitory factor) and indirect inhibition of parietal cell function as a result of changes in cytokines as well as hormonal, paracrine, and neural regulatory mechanisms (Konturek et al., 2000). H. pylori itself inhibits human H⁺K⁺-ATPase, α-subunit gene expression (Göoz et al., 2000). It also elicits secretion of at least 2 cytokines, interleukin 1β and tumor necrosis factor-α, that directly inhibit parietal cell secretion (Beales and Calam, 2001).H. pylori activates CGRP sensory neurons coupled to stimulation of somatostatin and thus inhibition of gastrin.

Most patients chronically infected with H. pylori manifest a pangastritis and produce less than normal amounts of acid. (Derakhshan et al., 2006) Reduced acid secretion, at the onset, is thought to be due to functional inhibition of parietal cells by either products of HP itself or, more likely, products of the inflammatory process, (Saha et al., 2007); this is usually reversible upon eradication of the bug (Shimatini et al., 2005).

Approximately 10% to 15% of patients acutely infected with H. pylori have antral predominant inflammation. These patients, who are predisposed to duodenal ulcer, produce increased amounts of acid as a result of reduced antral somatostatin content and elevated basal and stimulated

gastrin secretion (Gillen et al., 1998). The mechanism by which somatostatin secretion is decreased is not known but may involve cytokines induced by the inflammation and/or the production of N-α methyl histamine, a selective H3-receptor agonist, produced by H. pylori(Zavros et al., 2002). One may speculate that the H3-receptor agonist could diffuse across the antral mucosa to interact with H3 receptors on antral somatostatin cells, causing inhibition of somatostatin secretion, and, thus, stimulation of gastrin secretion (Vuyyuru et al., 1995). Gastrin, in turn, stimulates histamine secretion from ECL cells leading to enhanced acid secretion. Both interleukin-8 and platelet-activating factor are up-regulated in H. pylori-infected mucosa and are capable of stimulating gastrin release from isolated rabbit and canine G cells(Beales and Calam, 2001).

 

• Non-steroidal anti-inflammatory drugs (NSAI Ds)

NSAIDs such as ibuprofen (Nurofen, Brufen), diclofenac (Voltarol) or naproxen (Naprosyn) are used for treating painful conditions while aspirin is widely prescribed to prevent heart attacks and strokes. Unfortunately, both interfere with the gastric and duodenal defences, increasing the risk of ulceration. Thus the structure of diclofenac sodiumis shown in figure 4.

Figure 4. Structure of Diclofenac sodium.

Source: (National Center for Biotechnology, 2014)

 

Mechanism of action

NSAIDs are known to induce ulcers by inhibiting prostaglandin synthetase in the cyclooxygenase pathway (Rainsford, 1987). Prostaglandins are found in many tissues including the stomach, where they play a vital protective role via stimulating the secretion of bicarbonate and mucus, maintaining mucosal blood flow and regulating mucosal cell turnover and repair (Hayllar and Bjarnason, 1995). Thus, the suppression of prostaglandin synthesis by NSAIDs results in increased susceptibility to mucosal injury and subsequently gastric ulceration. The pathogenesis of NSAIDs-induced gastric ulceration includes the NSAID blocking the activities of the cyclooxygenase enzymes (COX-1 and COX-2) hence leading to reduced mucus and bicarbonate secretion, decreased mucosal blood flow, impaired platelet aggregation, alteration of microvascular structures leading to epithelia damage, reduced angiogenesis, and increased leukocyte adherence (Wallace et al., 2000). Increased production of reactive oxygen species (ROS), increased lipid peroxidation, and neutrophil infiltration also play a role in oxidative mucosal damage by NSAIDs (Lamarque, 2004). Various mechanisms have been proposed for this cytotoxic action of NSAIDs, including the induction of osmotic lysis subsequent to trapping of charged NSAIDs with the epithelial cells and death of the epithelial cell subsequent to uncoupling of oxidative phosphorylation (Djahanguiri, 1969). NSAIDs can also reduce mucus and bicarbonate secretion, thus decreasing the effectiveness of the juxtamucosal pH gradient in protecting the epithelium (Davenport, 1967). Furthermore, NSAIDs disrupt the layer of surface-active phospholipids on the mucosal surface, independent of effects on prostaglandin synthesis. Such an action would render the mucosa less able to resist damage induced by luminal acid (Linuma et al., 1993).

 

• Alcohol (Ethanol)

Ethanol is considered a risk factor for developing gastric ulcers. It readily penetrates the gastric mucosa due to its ability to solubilize the protective mucous and expose the mucosa to the proteolytic and hydrolytic actions of hydrochloric acid and pepsin (Oates and Hakkinen, 1988), causing damage to the membrane (Sener et al., 2004). Moreover, alcohol stimulates acid secretion and reduces blood flow leading to microvascular injuries, through disruption of the vascular endothelium and facilitating vascular permeability; it also increases activity of xanthine oxidase. Ethanol also triggers imbalances in cellular antioxidant processes. The harmful effects of ethanol thus manifest either through direct generation of reactive metabolites, including free radical species that react with most of the cell components, therefore changing their structures and functions, or by contributing to other mechanisms that finally support oxidative damage (Nordmann, 1994). Ethanol also produces necrotic lesions in the gastric mucosa of animals by a direct toxic effect thereby reducing the secretion of bicarbonates and depleting gastric mucus production in animals (Marhuenda et al., 1993). Furthermore, ethanol-induced membrane damage is associated with increased permeability of the plasma membrane by sodium and water. It also produces massive intracellular accumulation of calcium, which represents a major step in the pathogenesis of gastric mucosal injury. This leads to cell death and exfoliation in the surface epithelium (Massignani et al., 2009). Also, ethanol-induced ulceration is linked to reduced mucosa microcirculation and to increased apoptosis(Hernandez-Munoz et al., 2000).

 

 

• Stress

Stimulation of gastric acid secretion has historically been considered another mechanism by which stress increases susceptibility to duodenal ulceration, and researchers have reported increases in acid secretion in association with psychosocial stressors, (Feldman et al., 1992) especially among patients with duodenal ulcer (Bresnick et al., 1993).

 

• Smoking

The literature reveals a strong positive correlation between cigarette smoking and the incidence of ulcer disease, mortality, complications, recurrences and delay in healing rates. Smoking prevalences of 84% and 86% have been reported among patients with duodenal ulcer perforation (Smedley et al., 1988) and smokers have a threefold higher mortality from peptic ulcer than non-smokers (Doll et al., 1994). Smokers are about two times more likely to develop ulcer disease than nonsmokers. Cigarette smoking may increase susceptibility, diminish the gastric mucosal defensive factors, or may provide a more favorable milieu for H. pylori infection.

 

1.2.4.3       Inhibitors of acid secretion

• Somatostatin

Somatostatin is synthesized from a 92-amino acidpreprosomatostatin precursor molecule that is processedto yield somatostatin-14 and somatostatin-28.  Somatostatin-28 is the major form in the small intestine. In stomach, somatostatin cells are closely coupled to their target cells (eg, parietal, ECL, and gastrin cells) either directly via cytoplasmic processes or indirectly via the local circulation (Schubert et al., 1985). In the stomach, the actions of somatostatin are thought to be mediated via the somatostatin subtype 2 receptor (Allen et al., 2002). Gastrin, GRP, VIP, PACAP, β2/β3-adrenergic agonists, secretin, ANP, adrenomedullin, amylin, adenosine, and CGRP stimulate somatostatin whereas acetylcholine and interferon-y inhibit somatostatin secretion. An increase in luminal acidity acts to attenuate acid secretion via a pathway involving release of somatostatin in both antrum and fundus.

 

• Other inhibitors

ANP, CCK, secretin, glucagon-like peptide, peptide YY, adrenomedullin, amylin, neurotensin, glucose-dependent insulinotropic polypeptide, leptin, and epidermal growth factor stimulate somatostatin and thus inhibit acid secretion (Hirsch et al., 2003). CCK may function as a physiologic enterogastrone, i.e. an intestinal factor responsible for the inhibition of acid secretion induced by the presence of nutrients in the intestine (Lloyd et al., 2001). Interleukin-1β and serotonin inhibit acid secretion (Beales and Calam, 2001).

 

1.2.5       Diagnosis and Prevention of Peptic Ulcer Disease

1.2.5.1    Noninvasive Techniques

Blood test: A blood sample is taken from the patient’s vein and tested for H. pylori antibodies. Antibodies are substances the body produces to fight invading harmful organisms called antigens such as the H. pylori bacterium.

 

¹⁴C Urea breath test: The breath tests are performed by having the patient swallow carbon-labelled urea which is metabolised by H. pylori.H. pyloriproduced urease to release labelled carbon dioxide. This is absorbed into the blood stream and then exhaled in the breath of infected individuals.
Procedure: The patient is given a ¹⁴C urea capsule to swallow after an overnight fast. A single breath sample is taken, by blowing through a straw into a bottle of liquid, 10 to 15 minutes later and measured for radioactive CO₂. ¹⁴C urea is radioactive, but the ingested urea is rapidly excreted and the radiation dosage from this test is very low (equal to one day’s background radiation).

 

Stool antigen test: The patient provides a stool sample, which is tested for H. pylori antigens.

 

Barium-X ray test: For an upper gastrointestinal series, the patient drinks a white chalky barium. The barium marks the esophagus, stomach, and duodenum and any ulcers show up clearly on an x ray. Sedation is not necessary for this procedure.

 

Antibody test: Patients infected with H. pylori have immunoglobulin antibodies to the organism. Tests for the detection of antibodies to H. pylori circulating in blood, or found in saliva, have excellent sensitivity and specificity of above 95% and are cheap and simple compared with invasive techniques (DeCross and Puera, 1992). Antibody assays in blood have measured IgG and IgA antibodies which have been shown to be specific for H. pylori and not other gram-negative organisms (Perez-Perez et al., 1988). IgG assays tend to have slightly higher sensitivity and specificity, and so anti-IgG methods tend to be favoured (Crabtree et al., 1991). The commercially available assays are of two sorts: either microtitre-plate assays for use in a laboratory, or near-patient testing devices. Both types of assay usually have a cut-off value set with control sera so that they differentiate patients with H. pylori infection from those who do not, rather than quantify the concentration of circulating anti-H. pylori immunoglobulin.

1.2.5.2 Invasive Techniques: If a patient has any alarm symptoms, the doctor orders an endoscopy or upper gastrointestinal (GI) series.

 

Endoscopy: Endoscopy is a medical procedure that uses an instrument called an endoscope.

Looking with an endoscope is different from using imaging tests, like x-rays and CT scans which can get the inside pictures ofthe body without putting tools or devices into it. The patient is lightly sedated. The doctor passes an endoscope which is a thin, lighted tube with a tiny camera on the end into the patient’s mouth and down the throat to the stomach and duodenum. With this tool, the doctor can closely examine the lining of the esophagus, stomach, and duodenum. Patients older than 55 years and those with alarm symptoms should be referred for prompt upper endoscopy. Esophagogastroduodenoscopy (EGD) is more sensitive and specific for peptic ulcer disease than upper gastrointestinal barium studies and allows biopsy of gastric lesions (Talley et al., 2005). If an ulcer is bleeding, the doctor can use the endoscope to inject medicines that help the blood clot or to guide a heat probe that burns tissue to stop bleeding. This process is called cauterization.

Culture test: H. pylori can be cultured only when a specimen containing the pathogen has been obtained, and in this case that means obtaining a biopsy specimen at endoscopy. Methods of culture vary, but in general they involve homogenising the biopsy specimen and culturing the homogenate on a variety of specialised agar plates at elevated temperatures for at least seven days.  For H. pylori, however, the success of the technique depends on local technique and access to facilities, and can be regarded as being not more than 60 – 90% sensitive, though being 100% specific; the cost of each test is high (DeCross and Puera, 1992; Perez-Perez et al., 1988).

 

Histology:This involves preparation of stained, thin (less than 5 micrometers, or 0.005 millimeters) slices mounted on a glass slide, under a very thin pane of glass called a coverslip. The fresh specimen is immersed in a fluid called a fixative for several hours (the necessary time dependent on the size of the specimen). The fixative, typically formalin (a 10% solution of formaldehyde gas in buffered water), causes the proteins in the cells to denature and become hard and “fixed.” Adequate fixation is probably the most important technical aspect of biopsy processing. The stomachs were sectioned and embedded in paraffin then stained with hematoxylin and eosin and then examined under a light microscope.

 

Table 2. Tests used in the diagnosis of Peptic Ulcer

Test                             Comments

EGD                            Indicated in patients with  evidence of bleeding, weight loss, chronicity, or persistent vomiting; those whose symptoms do not respond tomedications and those older than 55 years. More than 90 % sensitivity and specificity in diagnosing gastric and duodenal ulcers and cancer.   Barium and diatri        Indicated when endoscopy is unsuitable or not feasible,or if complications zoate sodium               such as gastric outlet obstruction suspected. contrast radiology       Diagnostic accuracy increases with extent of disease; 80 to 90% sensitivity in detecting duodenal ulcer.   H. pylori testing Serologic ELISA         Useful only for initial testing (sensitivity 85%; specificity, 79%); cannot be used to confirm eradification.   Urea breath test            More expensive (sensitivity, 95 to 100%; specificity,91 to 98%; can be used to confirm eradication. PPI should be stopped for two weeks before test.   Stool antigen test         Inconvenient but accurate (sensitivity, 91 to 98%; specificity, 94 to 99%) Can be used to confirm eradication.   Endoscopic biopsy       Culture (sensitivity, 70 t0 80%; specificity 100%), histology (sensitivity > 94%; specificity 100%).

Source:  Talley et al. (2005)

 

 

1.2.5.3   Prevention of Peptic Ulcer

• Learn which anti-inflammatory drugs that are safe to take. Acetaminophen is recommended over aspirin, naproxen, ibuprofen and other drugs NSAIDs.
• Reduce consumption of alcoholic and caffeinated beverages.
• Quit smoking and avoid second-hand smoke.
• Patients with peptic ulcers should avoid diets rich in red meat, fried or fatty foods, and refined foods, such as flour or sugar.
• Keeping meals small, and eating 5 to 6 times a day at regular intervals, can level out stomach acid.
• Take all prescribed medication.
• Reduce stress.

 

1.2.6    Classification of Antiulcer Agent

 

1. Reduction of gastric acid secretion e.g.

H2 antihistamines: Cimetidine, ranitidine, famotidine, roxaatidine, loxatidine.

PPI’s: Omeprazole, lansoprazole, pantoprazole, rabeprazole, esomeprazole.

Anticholinergics: Pirenzepine, propantheline, oxyphenonium.

Prostaglandine analogues: Misoprostol, enprostil, rioprostil.

 

1. Neutralization of gastric acid (Antacids) e.g.

 Systemic: Sodium bicarbonate, sodium citrate hydroxide.

 Nonsystemic (Local): Magnesium trisilicate, Aluminium hydroxide gel, Calcium carbonate (Goodman and Gilman’s, 2008).

1. Ulcer protectives: Sucralfate, CBS (Colloidal Bismuth Subcitrate).

d.Ulcer healing drugs: Carbenoxolone sodium (Tripathi, 2004).

1. Anti-H.pyloric drugs: Amoxicillin, Clarithromycin, Metronidazole, Tinidazole, Tetracycline.

 

• Histamine-H2 Receptor Antagonist

Histamine receptor is on parietal cells (autonomic nervous system). Food stimulates gastrin release, gastrin stimulates histamine release, histamine stimulates parietal cells secretion of HCl.

 

Mechanism of action

The H2 receptor antagonists inhibit acid production by reversibly competing with histamine for binding to H2 receptors on the basolateral membrane of parietal cells. They block histamine from stimulating the acid-secreting parietal cells of the stomach. H2 antagonists are mainly basal, psychic and neurogenic. Gastric secretion is suppressed and other stimuli such as Ach, gastrin, alcohol and food are also inhibited. (Goodman and Gilman’s, 2008)

 

Reversible competitive inhibitors of H2 receptor

They are highly selective for H2 receptors. They are very effective in inhibiting nocturnal acid secretion (as it depends largely on histamine). They have modest impact on meal stimulated acid secretion (as it depends on gastrin, acetylcholine and histamine).

 

Pharmacokinetics

They are absorbed rapidly and completely except for famotidine. Food and antiacids may reduce absorption; distributed widely throughout the body; metabolized by the liver; excreted primarily in the urine.

 

 

 

 

Therapeutic Uses

Used therapeutically to promote healing of duodenal and gastric ulcers.  They provide long-term treatment of pathological gastrointestinal hypersecretory conditions. They reduce gastric acid production and prevent stress ulcers.

Adverse effect

Headache, bowel upset and dizziness. Cimetidine causes gynecomastia, galactorrhea (as it is antiandrogenic and increases prolactin level).

Interaction

Inhibits CYP-450 which leads to the metabolism of many drugs so that they accumulate to toxic level. e.g. theophylline, warfarin, phenytoin, quinidine.

Contraindication

Renal impairment and hepatic failure.

 

• Proton Pump Inhibitors (PPIs)

They disrupt chemical binding in stomach cells to reduce acid production, lessening irritation and allowing peptic ulcers to heal. These drugs include: Rabeprazole, Omeprazole, Esomaprazole, Lansoprazole, Pantoprazole (Scanlon and Tina, 2007).

Mechanism of action

They block the last step in the secretion of gastric acid by combining with hydrogen, potassium, and adenosine triphosphate in the parietal cells of the stomach. They are most effective drugs in antiulcer therapy. They are irreversible inhibitor of H⁺K⁺– ATPase to apical membrane of the parietal cell. They are prodrugs requiring activation in acid environment. They are inactivate at neutral pH, at pH<5 rearranges to 2 charged cationic form sulphenic acid and sulphenamide. They react covalently with –SH groups of the H⁺K⁺– ATPase .They are weakly basic drugs and so accumulate in canaliculi of parietal cell. They are activated in canaliculi and binds covalently to extracellular domain of H⁺K⁺-ATPase.  Acid secretion resumes only after synthesis of new molecules.

Adverse effect

Nausea, dizziness, hepatic dysfunction, and headache.

 

Interaction

Inhibits oxidation of certain drugs such as Diazepam, Phenytoin, and Warfarin.

• Antacid

These are the basic substances which neutralize gastric acid. Acid neutralizing capacity is given as number of mEq of 1N HCl that brought to pH 3.5 in 15 minutes by a unit dose of the antacid preparation. Antacid might be systemic or non-systemic.

Systemic antacid:e.g. Sodium bicarbonate. It is water soluble. Its intravenous duration of action is short. It is a potent neutralizer, pH may rises above 7. It produces CO₂ in stomach which leads to distention and discomfort. It increases sodium load as such worsen CHF with edema. Large dose produces alkalosis.

Non-systemic antacid: These are insoluble and poorly absorbed basic compound. They react in stomach with acid to form respective chloride salts. This again reacts with bicarbonate and is not spared for absorption, hence no acid –base disturbance. e.g. Aluminium hydroxide gel.  The Al³⁺ ions relaxes smooth muscle which leads to delay in gastric emptying. This causes constipation. Mucosal astringent reaction also leads to constipation (Scanlon and Tina, 2007).

 

• Ulcer Protectives e.g. Sucralfate

Sucralfate consists of the octasulfate of sucrose to which Al(OH)³ has been added.  In an acid environment(pH <4), sucralfate undergoes extensive cross-linking to produce a viscous, sticky polymer that adheres to epithelial cells and ulcer craters for up to 6 hours after a single dose. In addition to inhibiting hydrolysis of mucosal proteins by pepsin, sucralfate may have additional cytoprotective effects, including stimulation of local production of prostaglandins and epidermal growth factor.  Since it is activated by acid, sucralfate should be taken on an empty stomach 1 hour before meals. The use of antacids within 30 minutes of a dose of sucralfate should be avoided. The usual dose of sucralfate is 1 g four times daily (for active duodenal ulcer) or 1 g twice daily (for maintenance therapy).

 

• Anti pylori drugs

They promote back diffusion of H⁺. Antimicrobials found clinically effective against H.pylori include Amoxicillin, Clarithromycin and Metronidazole. Single drug rapidly develops resistance (metronidazole). Colloidal bismuth subcitrate is active against H.pylori and resistance does not develop to it (Goodman and Gilman’s, 2008).

1.2.7     Treatment of Peptic Ulcer Disease

The rationale behind the treatment of peptic ulcer disease is twofold.  Firstly,  reduction of

hostile factors is essential, as is augmentation of protective factors. Antacids, histamine H2-receptor antagonists, proton pump inhibitors (e.g., omeprazole, lansoprazole), and surgery succeed by neutralization or reduction of gastric acid. Secondly, boosting mucosal protection  with drugs such as Sucralfate and prostaglandin.

 

Table 3. Treatment of Peptic Ulcers

Treatment                       Comment                  Options

Eradication of      Treatment duration is 10     Omeprazole 20 mg two times daily or lansoprazole H.pylori                to 14 days                             30 mg two times daily plus amoxicillin 1 g two times daily or metronidazole 500 mg two times daily (if allergic to penicillin) Eradication rates 80 to 90    plus clarithromycin 500 mg two times daily. Percent or higher                 Ranitidine bismuth citrate 400 mg two times daily. plus clarithromycin 500 mg two times daily or metronidazole 500 mg two times daily plus tetracycline 500 mg two times daily or amoxicillin 1 g two times daily. Levofloxacin 500 mg daily plus amoxicillin1 g two times daily plus pantoprazole 40 mg two times daily. Bismuth subsalicylate 525 mg (two tablets) four times daily. plus metronidazole 250 mg four times daily plus tetracycline 500 mg four times daily plus H2 blocker blocker for 14 days or PPI for 28days. Histamine H2         70 to 80 percent healing       Ranitidine 150 mg two times daily or 300 mg at Blockers              in duodenal ulcer after 4      night. weeks, 87 to 94 percent       Famotidine 20 mg two times daily or 40 mg at after 8 weeks                        night. Cimetidine 400 mg two times daily or 800 mg at night. Proton pump      Treatment duration is 4         Omeprazole 20 mg daily. inhibitor             weeks for duodenal ulcer       Lansoprazole 15 mg daily. and 8 weeks for gastric          Raberprazole 20 mg daily. ulcer 80 to 100 percent healing       Pantoprazole 40 mg daily. Sulcraphate        Treatment duration is 4          1 g four times daily. weeks. Effectiveness is similar to H2 blockers Surgery               Rarely needed                        Duodenal ulcer: truncal ulcer, selective ulcer, highly selective vasotomy,partial gastrectomy. Gastric ulcer: partial gastrectomy with gastroduodenal or gastrojejunal anastomosis

Source: (Behrman, 2005)

Medical Therapy

The recommended duration of therapy for eradication is 10 to 14 days. However, shorter treatment courses (regimens of one, five, and seven days) are being assessed. (Lara et al., 2003)Administration of a H2 blocker or proton pump inhibitor for four weeks (Table 3)induces healing in most duodenal ulcers. Proton pump inhibitors provide superior acid suppression, healing rates, and symptom relief and are recommended as initial therapy for most patients.Repeated EGD with biopsy is recommended to confirm healing of gastric ulcers and to rule out malignancy.

 

Surgery

Patients who are intolerant of medications or who do not comply with medication regimes, and those at high risk of complications (e.g., transplant recipients, patients dependent on steroids or NSAIDs, those with giant gastric or duodenal ulcer, those with ulcers that fail to heal with adequate treatment) are referred to surgery. Surgery should also be considered for patients who have a relapse during maintenance treatment or who have had multiple courses of medications(Palanivelu et al., 2003).

1.3 Gastric Content Parameters

These are parameters that determine the extent of gastric lesions that occurred in epithelial linings of the gastric walls of the stomach. These parameters include:

 

1.3.1Ulcer Index

The term ulcer index means the number of ulcers in the gastric walls which is caused by the secretion of strong acid (HCL) and enzymes (pepsin) by the parietal and chief cells respectively. After sacrificing the rats, the stomach were removed and opened along the greater curvature.  The severity of hemorrhagic erosions in the acid secreting glandular mucosa were assessed on a scale of 0 to 3.

0= normal

1= one to four petechiae

2= five or more petechiae or hemorrhagic streaks up to 4 mm and

3= erosions longer than 5 mm or confluent haemorrhages (Maity, 1998).

 

1.3.2   Gastric volume/Gastric residual volume (GRV)

GRV was the primary determinant in slowing infusion rates (Payne et al., 1996).The most commonly reported GRV considered for a “cut-off” value was between 100 to 150 mL. Many clinicians believe or assume that there is tight correlation between aspiration of gastric contents and an elevated GRV. This assumption implies that when a cutoff value for GRV is increased, the aspiration of gastric contents will also increase.e

 

 1.3.3    Gastric pH

pH is equal to −log₁₀ of hydrogen ion concentration in moles per litre. Gastric pH is  the acidity of the gastric juice solution on a logarithmic scale on which 7 is neutral, lower values are more acid and higher values more alkaline. The pH of gastric acid is 1.5 to 3.5 (Marieb and Hoehn, 2010) in the human stomach lumen, the acidity being maintained by the proton pumpH^{HYPERLINK “http://en.wikipedia.org/wiki/Hydrogen_potassium_ATPase”+}HYPERLINK “http://en.wikipedia.org/wiki/Hydrogen_potassium_ATPase”/K^{HYPERLINK “http://en.wikipedia.org/wiki/Hydrogen_potassium_ATPase”+}HYPERLINK “http://en.wikipedia.org/wiki/Hydrogen_potassium_ATPase” ATPase. The parietal cell releases bicarbonate into the blood stream in the process, which causes a temporary rise of pH in the blood, known as alkaline tide. The lowest pH of the secreted acid is 0.8 (Guytonand John, 2006).

 

1.3.4    Total and Free Acidity

Total aciditycan be defined as the concentration of excess acid in a solution determined by the amount of strong base it is necessary to add to the solution in order to produce a given final state. The free acid value tells how much acid is available to initiate the reaction and exists in its original “active” state. This would involve taking a bath sample and titrating it with a known standard such as sodium hydroxide (probably 0.1N or 1.0 N). An indicator is added to the solution that changes color when the titration reaches a certain pH. A common indicator for this would be thymol blue which changes from reddish orange to orange yellow when passing through the pH range around 3.5-4.0. The first titration would be for free acid. The total acid value is meant to indicate the total amount of acid that has been put into the system and is a combination of both free acid and that which has been “neutralized” through the reaction. To determine total acid content, one would repeat most of the same steps above, until blue is recovered in the pH range of 8.5-9.0.

 

1.4      Biochemical Parameters

1.4.1   Platelet Count

A platelet count is a component of the complete blood count (CBC) or full blood count (FBC). The platelet count measures the number or concentration of platelets in the bloodstream. The normal platelet count is between 150 and 450 billion (or 10⁹) per liter of blood. In some cases the platelets are expressed as the number per cubic millimeter or microliter of blood, with the normal range being between 150,000 and 450,000. (James et al., 2004) Human platelets are small and discoid in shape, with dimensions of approximately 2.0–4.0 by 0.5lm, and a mean volume of 7–11 fl. (George, 2000) They are the second most numerous corpuscle in the blood normally circulating at between 150–450 x10⁹/l. Platelets are anucleated cells derived from megakaryocytes and typically circulate for 10 days. (George, 2000) Platelets are involved in many pathophysiological processes including haemostasis and thrombosis, clot retraction, vessel constriction and repair, inflammation including promotion of atherosclerosis, host defence and even tumour growth/metastasis. When there is a defect in any of these functions and/or platelet number, haemostasis is usually impaired and there may be an associated increased risk of bleeding. In contrast, a marked increase in platelet number or reactivity can lead to inappropriate thrombus formation.

 

1.4.2     Lipid profile

A lipid profile is a blood test that measures the amount of lipids, or fats, found in the blood. The lipids measured are usually total cholesterol, HDL cholesterol, LDLcholesterol and triglycerides. When levels of these lipids are abnormal, there is an increased risk of heart attack and stroke. Units for these are mg/dl, or milligram per deciliter. A deciliter is 1/10^th of a liter. Lipids generally included in a blood lipid profile are described below.

Cholesterol: Cholesterol is a necessary molecule in human metabolism. It is a component of cell membranes, and is a building block of bile, estrogen and testosterone. The cholesterol necessary for normal metabolism is synthesized by the liver. Generally, a level less than 200 mg/dl is considered desirable. Between 200 mg/dl and 240 mg/dl is considered borderline high, and over 240 mg/dl is considered high. Cholesterol is a lipid and is insoluble in water. It is transported through the blood encased in a soluble protein. Cholesterol is present in the blood in four forms.  Examples are total cholesterol, HDL-cholesterol, LDL-cholesterol and triglyceride. These fourforms are all combinations of protein, cholesterol and triglyceride (American Heart Association, 2005).

 

Triglycerides: This is the most common type of lipid found in animals. Fat tissue is primarily for the storage of this form of lipid. The body will convert any form of excess calories into triglycerides for long-term storage. A value below 150 mg/dl indicates no increased risk, 150 -200 indicates a slight risk, and over 200 mg/dl is a high risk(American Heart Association, 2005).

 

LDL Cholesterol, or Low density lipoprotein: This is sometimes referred to as the “bad cholesterol.” This form contains the highest amount of cholesterol. A value between 130 – 159 mg/dl is borderline high, and over 160 mg/dl is considered ‘high” (American Heart Association, 2005).

 

HDL Cholesterol, or High density lipoprotein: This is sometimes called “good cholesterol.” The higher the number, the better the protective effect against cardiovascular disease as it removes excess cholesterol from circulation and carries it back to the liver where it is degraded or converted into bile acids (Ahmad et al.,1992). A value below 40 mg/dl is considered a risk factor. A value above 60 mg/dl is considered protective against heart disease. HDL cholesterol is cholesterol that is packaged for delivery to the liver, where the cholesterol is removed from the body.

 

1.5   Aim and Objectives of the Study

 

1.5.1 Aim of the Study

The aim of this study was to investigate the anti-ulcer activity of aqueous tuber extract of Cyperus esculentus in diclofenac sodium and histamine -induced gastric lesions in Wistar

albino rats.

1.5.2   Specific Objectives of the Study

The aim of this study was achieved through the following specific objectives:

• To determine the possible lethal dose (acute toxicity) of the aqueous extract of Cyperus esculentustuber in ulcerogenic rats.
• To determine the ulcer index and percentage protection in ulcerogenic rats treated with the aqueous extract of Cyperus esculentus
• To determine the gastric volume in ulcerogenic rats treated with the aqueous extract of Cyperus esculentus
• To determine the gastric pH in ulcerogenic rats treated with the aqueous extract of Cyperus esculentus
• To determine the free acidity and total acidity in ulcerogenic rats treated with the aqueous extract of Cyperus esculentus
• To determine the lipid profile in ulcerogenic rats treated with the aqueous extract of Cyperus esculentus
• To determine the platelet count in ulcerogenic rats treated with the aqueous extract of Cyperus esculentus
• To determine the plasma glucose level in ulcerogenic rats treated with the aqueous extract of Cyperus esculentus
• To determine the in vitro effect of histamine on Wistar albino rats’ stomach treated with the aqueous extract of Cyperus esculentus

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