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ISOLATION AND IDENTIFICATION OF ANTI-DIABETIC AGENTS FROM SELECTED MEDICINAL PLANTS

CHAPTER ONE

INTRODUCTION

1.0 Introduction

Anti-diabetic agents are medications that are used to manage high blood sugar levels in people with diabetes (Cappon et al., 2019), There are several types of anti-diabetic agents, including; Insulin: Insulin is a hormone that is produced by the pancreas and helps the body use glucose (sugar) from food for energy (Dunlay, 2019)., 

1.1 Background of the study

Diabetes is a chronic disease that affects how somes’ body processes blood sugar (glucose), which is the primary source of energy for the cells (Foretz, Guigas, &Viollet, 2019),  Insulin is a hormone produced by the pancreas that regulates blood sugar levels, and in people with diabetes, the body either doesn’t produce enough insulin or doesn’t use it effectively (Glovaci et al., 2019), resulting in high blood sugar levels, there are several types of diabetes, including; Type 1 diabetes: This type of diabetes occurs when the immune system attacks and destroys the cellss in the pancreas that produce insulin. According to the International Diabetes Federation (IDF) Diabetes Atlas 9th edition, the prevalence of diabetes in Uganda among adults (20-79 years) was 1.6% in 2019. This translates to approximately 453,000 adults living with diabetes in the country. The IDF also estimated that there were an additional 200,000 cases of undiagnosed diabetes in Uganda in 2019.The prevalence of diabetes in Uganda is lower than the global average of 9.3%, but the burden of the disease is expected to increase in the coming years due to population growth, aging, and lifestyle changes. Diabetes is a major public health concern in Uganda, and there is a need for increased awareness, screening, and access to diabetes care and treatment in the country.

1.2 Problem statement

Diabetes is one of the top non-communicable diseases in Africa, contributing to the increasing disease burden among the old adults. In uganda, diabetes mellitus is a silent major non communicable disease and the T2DM is a major and growing problem in the country (Gatimu et al., 2016).. Diabetes mellitus is one of the still most important non-communicable disease and the T2DM is a key rising problem in the country. T2DM is a silent killer as several of the cases go undiagnosed (Darko, et al., 2021). High levels of ignorance of the populace about the disease is also a factor in Uganda. The high cost related to the diagnosis and treatment of diabetes has also been a main concern, the cost of treating diabetes by synthetic drugs is high and this has led to many patients dying in their homes unable to get treatment in the hospitals. This has prevented the individual from undergoing check-ups to find their status with respect to the disease as the cost is beyond numerous household incomes, Hence, there is a need to develop low-cost solutions for diabetes, especially in underserved areas in different regions from naturally occurring plants.

1.3 Objectives

1. Identify and collect medicinal plants with anti-diabetic agents used in treatment of diabetes.
2. Testing the crude extracts for anti-diabetic activity 
3. Isolation and purification of the Bio-active compounds in the extracts
4. Structure elucidation of the Bioactive compound in the extract with anti-diabetic properties

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-diabetic activity. 

The following methods will be used to identify plants with anti-diabetic agent and they include; Traditional knowledge (Al-Megrin et al., 2020).

Botanical identification. 

Chemical identification.

Pharmacological testing.

3.1.1 Procedure to identify and authenticate plants for experiments

Taxonomic identification: The first step is to accurately identify the plant species. This can be done through taxonomic identification.

The process of taxonomic identification typically involves several steps; Collecting the specimen: The first step is to collect the specimen for identification.

Comparison with existing taxonomic keys.

Naming the organism.

Voucher specimens.

Authentication

Quality control.

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

To test crude extracts for anti-diabetic 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. 

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.

3.2.2 Fractionation of Hydro-Methanol Extract

n-Hexane, chloroform, and water are to be used as solvents for fractionation.

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) (Jain et al., 2014).

3.2.5. Grouping and Dosing of Animals

Inducing of experimental diabetes  in rats.

Acute Hypoglycemic Effect.

Hyperglycemic animals are to be divided into seven groups of six rats each.  Group 1, is to be used as control group, is to be treated  orally with   1.5 ml of a physiological NaCl solution (EsF., Andrade-Cetto, A., Escandón-Rivera, S., Mata-Torres, G., & Mata, R. (2021).).

Maltose and Sucrose Tolerance Tests.

 Hyperglycemic animals are to be given seven groups containing four rats each.

2.5. Crude Small Intestine Extract. 

2.6. In Vitro Glucosidase Assay. 

2.7. Statistical Methods. 

The data is to be analyzed using an ANOVA test, followed by Fisher’s post hoc test using the software Statistician. The plasma glucose levels are then expressed as the mean (SEM); significance considered at least with p< 0.05.

3.3 Isolation and purification of the bio-active compounds in the extracts

Analytical HPLC

Procedure

Analytical HPLC will be employed for method development for small scale separation and purity

assessment of isolated compounds. 

3.3 Structure elucidation of the bioactive compound in the extract with anti-diabetics properties

Spectroscopic methods

For structural characterization of the isolated compounds, principally NMR and mass spectrometry will be performed. UV and IR spectroscopy along with melting point determination, and optical rotation analysis will also be carried out when required for physical characterization and structural elucidation of isolated compounds (Mortazavi et al., 2020).

UV-visible spectroscopy

UV spectroscopy will be used for the preliminary identification of compounds, particularly for the

identification of the presence of phenolic groups or conjugated double bonds (Mohammed, 2019).

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