EVALUATION OF THE GASTROPROTECTIVE AND SUB-CHRONIC TOXICITY OF METHANOL STEM BARK EXTRACT OF XIMENIA AMERICANA LINN (OLACACEAE) IN MALE WISTAR RATS

Abstract

Ximenia americana is a plant used traditionally in northern parts of Nigeria for treatment of leprotic ulcers and mouth ulcers in addition to other medicinal uses. It contains flavanoids, saponins, tannins among others that has been reported to possess gastroprotective potentials. . The aim of this study is to evaluate the gastro-protective effects and establish the toxicity profile of the methanol stem back extract of Ximenia americana. The oral median lethal dose of the extract was estimated in mice and rats using Lorke‟s method of acute toxicity test, while the composite constituents of the extract were determined using established standard preliminary phytochemical screening procedures of colour changes in test-tube chemical reactions. The antiulcer effect of the extract was evaluated using two ulcerogenic models of indomethacin and ethanol induced ulcers in rats at extract doses of 250, 500 and 1000 mg/kg body weight. Sub-chronic toxicities of the extract on some organs, haematological and biochemical parameters were also investigated following 28 days daily pretreatment in rats. Alkaloids, anthraquinones, carbohydrates, flavonoids, saponins, steroidal glycosides, tannins and terpenoids were found to be present in the methanol stem back extract of Ximenia americana. The extract caused no death in mice and rats at doses of up to 5,000 mg/kg oral administration and no observable behavioural changes were seen in both animal species (mice and rats) within 24 hours. The indomethacine-induced ulcer lesions were reduced in the extract pretreated groups of rats and in a dose dependent manner that was significant (p<0.05) for the two higher doses of the extract (500 and 1000 mg/kg) when compared to the control group. The extract significantly (p≤0.05) and dose-dependently reduced the mean ulcer spots in a similar manner as the standard agents cimetidine and misoprostol. The reduction was significant for only the two higher extract doses (500 and 1000 mg/kg). However, the ulcer lesions were more prevented in the rat groups of the standard drugs. A mean of only 3 severe ulcer spots for each of the two lower extract dose pre-treated rat groups against 9 severe ulcer spots for the normal saline control group occurred with the indomethacin ulcerogen. However, no severe ulcer spots were found in both the cimetidine and 1000 mg/kg extract pretreated rat groups. There were no severe ulcers (≥ 3mm spots) found with the ethanol-ulcerogen in any of the groups including the normal saline control group. In conclusion the extract did not cause changes in the organ sizes in relation to the control experiment. The body weights of the extract-treated rats did also not change. However, histological examinations showed vascular congestion with polymorphonuclear cells in the kidney at the extract dose of 1000 mg/kg; consolidated areas of polymorphs infiltration at the alveoli and terminal bronchioles of the lungs; and distorted germinal centres in spleen. No remarkable changes were observed of the heart, liver, stomach and brain. Amongst the evaluated electrolytes, only Na+, Cl- and Ca2+ showed consistent but slight increases in their concentrations across the various doses of the extract, while changes in K+ and HCO3- levels were neither significant nor of a consistent pattern. The liver enzymes including the aminotransferases and alkaline phosphatase were not altered. A dose dependent increase in total protein that was significant (p<0.05) only at 1000 mg/kg; and a reduction in albumin level that was significant at the two higher extract doses (500 and 1000 mg/kg) were observed. The changes in the urea level were inconsistent and insignificant, while the dose dependent reduction in creatinine level seen in this study was not significant at any of the doses. In conclusion, the methanol stem back extract of Ximenia americana possess gastro-mucosal protective properties that prevented ulceration and/or promoted ulcer healing and is also relatively systemically non-toxic.

CHAPTER ONE

1.0 INTRODUCTION

1.1 Treatment of Peptic Ulcer Disease (PUD)

Peptic ulcers are parietal painful sores that occur in the epithelial lining of the upper gut (stomach, esophagus, or small intestine) due to excess gastric acid secretion (Abbas and Kumar, 2010). The development of peptic ulcer of the oesophagus (reflux of stomach acid) is rare, thus the term peptic ulcer most commonly refers to gastric and duodenal ulcers. The major difference for both ulcers is the location of where the ulcer occurs, otherwise, the risk factors, possible causes, symptoms and diagnosis are often the same. However, duodenal ulcer appears to be less harmful, but the most common type peptic ulcer (Guyton and Hall, 2006). Stomach ulcer is more harmful and if improperly treated often results in life-threatening cancerous complications of medical emergency and hospitalisations that may require gastric surgery, including abnormal bleeding, perforative holes in the walls of stomach and duodenum and/or gastric outlet obstruction (Bardhan and Royston, 2014).

The three major factors that trigger over production of acid fluid and cause peptic ulcer are Helicobacter pylori (H-pylori) infection, excessive consumption of NSAIDS and gut problems like gastrinomas (Kawamura et al., 2013).

Pain and vomiting are particularly, the evident symptoms and may occur either singly or concurrently (Malfertheiner et al., 2009). Aside drug treatment to eradicate the underlying causative H. pylori and other offensive gastric mucosal microbial organisms, drugs are also used to either suppress acid secretion or increase mucosal resistance and protection and to enhance healing of ulcer wounds and/or relief symptoms (especially pain) (Anand, 2017).

The available synthetic drugs include gastric acid output inhibitors such as proton pump inhibitors [PPIs] like omeprazole or H2-receptor antagonists like cimetidine, anti-secretory agents such as prostaglandin analogues like misoprostol, and mucosal cytoprotectors e.g. sucralfate (Fashner and Gitu 2015). Thus, there is a quest for discovering more drugs with these activities, but with better efficacy and toxicity profiles.

1.2 Statement of Research Problem

The complications of gastric ulcer are life-threatening and are of medical emergency requiring hospitalizations. The mortality rate from PUD complications is more than 10 times that of other digestive diseases with perforation causing the highest mortality worldwide (Sung et al., 2010; Chung and Shelat, 2017).

In Africa, the incidence showed limited evidence that it is more common in urban areas and that its incidence is increasing further. However the highest prevalence in south part of Sub-saharan African and in the Nile-Congo watershed including Rwanda, Burundi and eastern Zaire of about 5-12%, a Tanzanian endoscopic finding revealed peptic ulcer incidence was 24.1% among adult patients with dyspepsia is of great concern (Tovey and Tunshall, 1975; Balint, 1989; Ayana et al., 2014).

A 12 year retrospective study of ulcer complication in Zaria, Nigeria as at 1998 showed that perforation had an increasing frequency of occurrence (45%) followed by gastric outlet obstructions (41%) (Ameh and Nmadi, 1998). As at 2005, a 13-year review at a Nigerian hospital found obstruction to be the most common complication (56%), followed by perforation (30%) (Irabor, 2005).

The high incidence of gastric ulcer complications in older subjects of above 60 years that had been reported to be reflecting increased use of NSAIDs is a major concern considering the inevitable need of these drugs (Sung et al., 2010; Liu et al., 2008). It has been reported that about 10% of peptic ulcer cases are NSAIDs induced (Bhala et al., 2013).

Gastric ulcer if improperly treated or left untreated results in complications and the use of the current antiulcer agents is often hampered by one adverse effect or the other; for instance:

Misoprostol has dose-related diarrhoea as common side effect and it suppresses gastric acid only at high doses. Thus, the consequent diarrhoeal effect impairs therapy compliance and/or militates against its frequent prescription. It also increases uterine tone and contractions and may cause uterine rupture, abortions or birth defects and thus, is contraindicated for pregnant women with gastric ulcers (Pasturak, 1998).

Misoprostol, a synthetic antisecretory agent of prostaglandin E1 analogue, is highly esteemed and approved for use in the prevention of NSAIDs-induced gastric ulcers. Thus, it is mostly indicated for subjects on NSAIDs that are at high risk of NSAID-induced ulcers, especially the elderly and people with ulcer complications (More, 2002).

The FDA had also disapproved the use of sucralfate, a complex of sucrose sulfate-aluminium hydroxide, probably due to its aluminium content-related astringent properties including nausea, vomiting and constipation; as well as risk of aluminium toxicity from accumulation, hypophosphataemia from depletion of the phosphate moieties and/or drug interactions that impair concurrent use with other drugs (Joseph et al., 2000; Fashner and Gitu, 2015).

1.3 Justification for the Study

Although natural products are not devoid of toxic side effects there is need for continuous search for efficacious agents of natural origin with minimal toxicity, at least in part, because they are readily available and at reduced cost.

The written record of the use of plants and herbs to maintain health and cure many ailments dates back to several years (Petrovska, 2012). Most developing and 3rd world countries resort to herbal forms of treatment because of the inaccessibility of conventional health care systems, unavailability or high cost of orthodox or synthetic drugs (WHO, 2012; Ekor, 2013).

WHO had estimated that approximately 80% of the rural dwellers and up to 80% of the world‟s population rely on herbal medicines as alternative options for their primary health care needs (WHO, 2012).

The ethnomedicinal claims of use of the various parts of Ximenia americana in Africa for spasmodic bowel diseases and ulcers amongst other ailments require scientific validation.

1.4 Aim and Objectives

The aim of this research work is to evaluate the gastroprotective properties and toxicity profile of the methanol stem back extract of Ximenia americana.

The Specific Objectives are as follows:

i. To determine the phytochemical constituents of the methanol stem bark extract of Ximenia americana.

ii. To establish sub-chronic toxicity profile of the methanol stem bark extract of Ximenia americana and determine its oral LD50 in mice and rats.

iii. To evaluate the gastro-mucosal protective effect of the methanol stem bark extract of Ximenia americana.

1.5 Research Hypothesis

The methanol stem bark extract of Ximenia americana possesses significant gastroprotective effect on gastromucosal linings from gastric acid erosion and is relatively safe.