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ISOLATION AND IDENTIFICATION OF ANTI-ULCER AGENTS FROM THE EXTRACTS OF SELECTED MEDICINAL PLANTS

CHAPTER ONE

INTRODUCTION

1.0 Introduction

Anti-ulcer agents are medications that are used to treat and prevent ulcers, which are sores that form on the lining of the stomach or upper part of the small intestine.

1.1 Background of the study

Peptic ulcer disease (PUD), a common disorder of the digestive system, is defined as digestive tract injury that results in a mucosal break greater than 3–5 mm (Fang et al., 2019), with a visible depth reaching the submucosa (Xie, et al., 2022), Mainly occurring in the stomach and proximal duodenum (Fang, 2019).

Proton pump inhibitors (PPIs): These drugs reduce the amount of acid produced in the stomach and help to relieve pain and promote healing of the ulcer. PPIs are commonly used in combination with antibiotics to treat H. pylori infection, H2 blockers: These drugs also reduce the amount of acid produced in the stomach and help to relieve pain and promote healing of the ulcer, Antacids: These medications neutralize stomach acid and can provide temporary relief of symptoms (Kowada, & Asaka, 2022).

Because of their cost, accessibility, desire for individualized health care, and fear of the negative effects associated with synthetic drugs, natural goods have remained the drugs of choice for some people due to their safety and efficacy. Also, usage increases as new infectious diseases emerge and conventional medications fail to treat diseases like cancer (Akinwumi, & Sonibare, 2019). In affluent nations, 80% of people utilize traditional remedies made from plants, whereas more than 30% of pharmaceuticals now have some connection to plants, either directly or indirectly. Out of the 252 drugs on the World Health Organization’s (WHO) list of essential medicines, 11% are entirely plant-based, and 122 plant-derived drugs have 80% of their uses related to their original ethnopharmacological purposes. It is estimated that 25% of all drugs prescribed globally are derived from plants (Kuna, et al., 2019).

1.2 Statement of the problem

Peptic ulcers are a type of sore that forms in the lining of the stomach, duodenum (the first part of the small intestine), or esophagus (Bereda, 2022). They are commonly caused by a bacterial infection with Helicobacter pylori or prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) (Dore, & Pes, 2021). In severe cases, complications such as bleeding or perforation of the stomach or duodenal wall can occur, which can be life-threatening. Peptic ulcers also have a significant economic burden, for both people living in developed and developing countries, with the cost of treatment and lost productivity from time off work (Petta et al., 2019).

Herbal medicine can be important in treating peptic ulcers for several reasons, including; Fewer side effects: Herbal medicines are often considered to have fewer side effects than synthetic drugs used to treat peptic ulcers (Kuna et al., 2019). This can help to address the underlying causes of peptic ulcers and promote overall health and wellbeing. In summary, herbal medicine can be an important and effective tool in treating peptic ulcers, offering a range of benefits that can help to alleviate symptoms and promote healing (Bi, & Man, 2014).

1.3 Purpose of the study

The purpose of the study is isolation and identification of anti-ulcer agents from the extracts of selected medicinal plants

1.4 Objectives

Identify and collect medicinal plants with anti-ulcer agents used in treatment of peptic ulcers.
Testing the crude extracts for anti-ulcer activity

Isolation and purification of the Bio-active compounds in the extracts

Structure elucidation of the Bioactive compound in the extract with anti-ulcer properties

CHAPTER TWO

LITERATURE REVIEW

2.0 Literature Review

This section presents the isolation and identification of anti-ulcer agents from the extracts of selected medicinal plants in line to the study objectives;

2.1.1 Prevalence

The lifetime risk for developing a peptic ulcer is approximately 10% (Hein et al, 2017), Globally, as of 2010, approximately 250,000 people died of peptic ulcer disease down from 320,000 in 1990 (Rickard, 2022)., while by 2020 Peptic ulcer disease (PUD) affected more than four million people worldwide annually and has an estimated lifetime prevalence of 5−10% in the general population (Kang et al, 2011)

Worldwide, there are significant heterogeneities in coping approaches of healthcare systems with PUD in terms of prevention, diagnosis, treatment, and follow-up (Byun et al., 2020), Prevention is positively correlated with the development of infrastructures and the effectiveness of healthcare systems, the choice of diagnostic test and treatment approaches mainly relies on accessibility and cost, (Sonnenberg, & Genta, 2020), Therefore, quantifying and benchmarking health systems’ performance is crucial yet challenging to provide a clearer picture of the potential global inequities in the quality of care.

2.1.2 Peptic ulcers disease

PUD accounts for an estimated lifetime prevalence of 5–10% and an annual incidence of 0.1–0.3% in the general population in Western countries. Due to nonspecific symptoms, PUD assessment and treatment requires clinical caution due to severe complications such as bleeding, perforation, penetration into adjacent organs and gastrointestinal obstruction, all of which could require acute endoscopic or surgical treatment (Bereda, 2022).

2.1.3 Medicinal plants with anti-ulcer properties

All around the world, traditional green vegetables are an excellent and affordable source of nourishment for a balanced diet. These vegetables also function as traditional medicines for ailments like toothache (Amaranthus viridis L.), acute abdominal pain (Celosia argentia L.), painful urination (Portulacaoleracea L.), headache (Smithia sensitiva Ait. ), diarrhea (C. mimosoides L.), rheumatism and cough (Marsileaminuta Linn), and helminthes infestation (Spinaciaoleracea Linn.) (Kuna et al., 2019).

2.2 Isolation and purification of the Bio-active compounds in the extracts

Isolation and purification of bioactive compounds from extracts can be a complex process, but there are some general steps that can be followed. The specific techniques used will depend on the nature of the compounds and the properties of the extract. Here is a general overview of the steps involved in isolating and purifying bioactive compounds:

CHAPTER THREE

MATERIAL AND METHODS

3.0 Introduction

This section presents the study in line with the materials and methods to be used in line with study objectives.

3.1 Identification and collection of medicinal plants with anti-ulcer agents used in treatment of peptic ulcers.

The following methods will be used to identify plants with anti-ulcer agent and they include; Traditional knowledge: This involves relying on the knowledge and experience of indigenous or traditional communities who have been using the plant for medicinal purposes for generations. Botanical identification: This involves identifying the plant based on its physical characteristics, such as the shape and arrangement of leaves, flowers, stems, and roots. This can be done through careful observation, comparison with plant identification keys, or consultation with a botanical expert.

Chemical identification: This involves analyzing the chemical composition of the plant to identify its active compounds.

3.1.1 Procedure to identify and authenticate plants for experiments

The procedure to identify and authenticate plants for experiments typically involves several steps, including; Taxonomic identification: The first step is to accurately identify the plant species.

3.2 Objective: Testing the crude extracts for anti-ulcer activity

To test crude extracts for anti-ulcer activity, the following steps will be taken:

3.2.1 Experimental Animals

Swiss albino mice (20-30 g) and Wistar rats (150–250 g) of either sex will be used in this experiment. The laboratory animals will be obtained from the Faculty of veterinary medicine Makerere university. Animals will be contained in standard cages at room temperature and 12 hours of light and dark cycles, and will be acclimatized for one week before the study to the laboratory conditions. The animals will be fed with a standard pellet and tap water ad libitum. The protocol for use of animals will be undertaken as per guidelines for use of laboratory animals (Polo, et al., 2012).

3.2.1 Preparation of Hydro-Methanolic crude Extract

The leaves of the selected medicinal plants are to be washed with distilled water to remove dirt and dust. The cleaned plant materials will be dried at room temperature (Fentahun et al., 2017).

3.2.2 Fractionation of Hydro-Methanol Extract

n-Hexane, chloroform, and water are to be used as solvents for fractionation. Briefly, distilled water will be added into the crude extract of selected medicinal plant and dissolved by using a separating funnel.

3.2.3 Phytochemical Screening

Preliminary phytochemical screening tests will be carried out to determine the major classes of phytochemicals on the hydro-methanol extract of plants stem bark by using different standard test procedures.

3.2.4 Acute Toxicity Test

Acute toxicity study will be carried out using the guidelines described by the Organization for Economic Cooperation and Development (OECD) guideline No. 425. Single female Swiss albino mice fasted for four hours on the first day of the test then; 2000 mg/kg of the extract will be given by oral route using oral gavage and the mice will be observed for the manifestation of behavioral and physical changes and special attention will be given during the first four hours. Depend on the results from the first mice, the next 4 females’ animals will be employed and fasted for about four hours and then a single dose of 2000 mg/kg of the extract will be given orally and followed firmly in the same manner. The observation will be sustained daily for a total of fourteen days (Adane, Atnafie, Kifle, & Ambikar, 2021).

3.2.5. Grouping and Dosing of Animals

3.2.5.1 Pylorus Ligation-Induced Ulcer Model

The shay et al. model will be used with slight modification. Rats will be randomly divided into five study groups, each consisting of six animals. Group I will be the negative control (NC), which will receive a vehicle only (distilled water+6% Tween 80). Group II will be served as a positive control and rats will be pretreated with ranitidine 50 mg/kg for ten days. Groups III, IV, and V will be received 100, 200, and 400 mg/kg of hydro-methanolic extracts of plant extracts (Meng, 2019).

Rats will be fasted for 24 hours before the study but has free access to water till the last 4 hours. After 1 hour of the last drug treatment, animals will be anesthetized with diethyl ether and the abdomen will be opened by a small midline incision below the xiphoid process. The pyloric portion of the stomach will be lifted out and ligated carefully to avoid traction to the pylorus or damage to the blood supply of gastric mucosa. The stomach will be replaced carefully and the abdominal wall will be closed by interrupted sutures. After four hours of pyloric ligation, rats will be sacrificed by inhalational anesthetic ether. The abdomen will be opened, the cardiac end of the stomach will be dissected out and the content will be drained into a test tube. The gastric juice will be collected and centrifuged at 1000 rpm for 10 minutes the volume of the supernatant will be noted and taken for the determination of total acidity and pH. The stomach mucosa of each animal will be washed with saline and running water, labeled, and placed on sodium phosphate-buffered 10% formalin until it will be examined for lesions by using a hand lens (10X) and scored accordingly.

3.2.6 Ethanol-Induced Ulcer Model

The ulcer will be induced by administering ethanol following the method by Hollander et al., and previous studies to determine the antiulcer effect of repeated and single-dose administration. Animals will be randomly assigned to 10 groups each consisting of six animals. Group I and VI will receive the vehicle (distilled water+4% Tween 80 for single and repeated dose, respectively) and considered as NCs, whereas group II and VII will be served as a reference standard and pretreated with misoprostol 5 μg/kg (single and repeated for 10 days, respectively).

3.2.7. Indomethacin-Induced Ulcer Model

The ulcer will be induced with indomethacin at a dose of 18 mg/kg to evaluate the ulcer healing effect of the plant extract which will be compared with the NC (vehicle) and positive (misoprostol 5 μg/kg) treated groups. The treatment groups will receive 100, 200, 400 mg/kg (once daily). The first dose will be given 6 hours after induction of ulcer with indomethacin (18 mg/kg). Four days after ulcer induction, analysis will be done.

3.2.8. Parameters for the Evaluation of an Antiulcer Activity

Macroscopic Evaluation of the Stomach

The stomach will be opened along the greater curvature, rinsed with saline and running water to remove gastric contents and blood clots; then, the mucosa of each animal will be labeled and placed on sodium phosphate-buffered 10% formalin until it will be examined by a 10x magnifier lens to assess the formation of ulcers. The numbers of ulcers will be counted. The Kulkarni method will be used for scoring the ulcer as follows: normal colored stomach (0), red coloration (0.5), spot ulcer (1), hemorrhagic streak (1.5), deep Ulcers (2), and perforation (3).

3.2.9 Determination of pH and Gastric Volume

An aliquot of gastric juice will be taken and the pH of the solution will be measured using a pH meter based on the method of Tan . The volume of gastric juice of each animal will be measured after centrifugation with 1000 rpm for 10 minutes and analyzed since it is one parameter for the study of the antisecretory effect of the plant extract.

3.2.10. Determination of Total Acidity

An aliquot of 1 ml gastric juice diluted with 9 ml of distilled water will be taken and two drops of phenolphthalein indicator will be added. Then, it will be titrated with 0.01 N NaOH until a permanent pink color will be observed.

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