ABSTRACT
Gastric ulcers are a common cause of morbidity and mortality. A gastric ulcer is defined as a peptic ulcer restricted to the gastric mucosa, induced by exposure to excessive hydrochloric acid as well as excessive pepsin activity. It leads to impaired gastric function and pain. Conventional drugs used to manage this condition have toxicity as a common side effect leading to various undesirable effects, which appear to limit the use of these drugs thereby activating research for alternative drugs. African nightshades (Solanum nigrum L.) has been used traditionally as a herbal cure for gastric ulcers in different parts of the world. The objective of this study was to analyze, through morphologic and morphometric means, the anti-ulcerogenic activity of three S. nigrum genotypes namely, S. scabrum, S. sarrachoides and S. villosum genotypes found in Kenya on the rat stomach. Aqueous extracts of the three S. nigrum genotypes were obtained at the vegetative stage and subjected to phytochemical screening. The crude extracts were then administered to three groups of Wistar rats 30 minutes before administration of 1ml ethanol to induce ulceration. A negative control group was given distilled water orally, while a positive control group was given 1ml ethanol orally. Tissues were harvested from the stomach antrum and examined grossly, then processed for examination under light microscopy using Hematoxylin and Eosin, Periodic Acid Schiff and Masson’s Trichrome staining methods. The phytochemical testing revealed the presence of: terpenoids, tannins, saponins, flavonoids as well as glycosides. The three S. nigrum genotypes exhibited antiulcerogenic effects. S. scabrum showed the highest ulcer inhibition score of 76.4%, followed by S. sarrachoides with 72.5% and S. villosum with 63.3%. S. nigrum pretreated rats showed less gastric mucosal surface erosion, congestion, edema and hemorrhage. Penetrating ulcers in S. nigrum pretreated rats affected only the gastric pit region of the stomach mucosa, except for those of the S. villosum pretreated rats, which penetrated to affect the gastric glands. S. nigrum pretreatment resulted in more intense staining, (compared with rats that were not treated before ulcer induction) of the mucus regions of gastric glands with PAS and of inter- glandular connective tissue with Masson’s trichrome denoting less gastric damage in these animals. Microscopic ulcer index scores decreased 5.1, 3.6 and 2.4- fold in S. scabrum, S. sacharroides and S. villosum pretreated rats respectively. Results of this work show that extracts of the three S. nigrum genotypes are antiulcerogenic in varying degrees with S. scabrum being the most effective. The observed differences in ulcer inhibition capacities of the three S. nigrum genotypes may be attributed to genetic factors, which reportedly influence the nature and composition of bioactive ingredients synthesized in medicinal plants. Further studies to isolate and quantify phytochemicals responsible for this activity at different stages of plant maturity are recommended.
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
TABLE OF CONTENTS v
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS x
LIST OF APPENDICES xii
PUBLICATION xiii
ABSTRACT xiv
CHAPTER ONE
1 INTRODUCTION
1.1 Objectives 3
1.1.1 Broad objective 3
1.1.2 Specific objectives 3
1.2 Null hypothesis 3
CHAPTER TWO
2 LITERATURE REVIEW
2.1 Gastric ulcers 4
2.2 Treatment of gastric ulcers 5
2.3 Use of herbaceous plants for treatment of ailments 6
2.4 The stomach 8
2.4.1 General histological structure of the stomach 8
2.4.2 The non-glandular region 9
2.4.3 The glandular region 9
2.4.4 The cardiac gland region 10
2.4.5 The fundic gland region 11
2.4.6 The pyloric gland region 12
2.5 The rat stomach 12
2.6 Physiology of gastroprotection 13
2.7 Induction of ulcers in experimental animals 14
CHAPTER THREE
3 MATERIALS AND METHODS
3.1 Plant material collection and extract preparation 15
3.2 Sample preparation 15
3.3 Phytochemical analysis 16
3.4 Morphological determination of differences in anti-ulcerogenic activity of the three S.nigrum
genotypes on the rat stomach 18
3.4.1 Experimental animals 18
3.4.2 Acute toxicity studies 18
3.4.3 Drug administration 18
3.5 Morphometric determination of differences in anti-ulcerogenic activity of the three S. nigrum
genotypes on the rat stomach 20
3.5.1 Macroscopic morphometry 20
3.5.2 Microscopic morphometry 21
3.6 Occupational health 22
3.7 Safe disposal of carcasses, sharps and chemicals 22
3.8 Data analysis 23
CHAPTER FOUR
4 RESULTS
4.1 Phytochemical composition of plant extracts 24
4.2 Acute toxicity findings 25
4.3 Morphologic findings 26
4.3.1 Macroscopic findings 26
4.3.2 Light Microscopic findings 30
4.4 Morphometric findings 40
4.4.1 Macroscopic ulcer index 40
4.4.2 Microscopic ulcer index 40
CHAPTER FIVE
5 DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 Discussion 42
5.2 Conclusion 47
5.3 Recommendations 49
REFERENCES 50
APPENDICES 67
Appendix 1: Hematoxylin and Eosin (H & E) Staining Protocol 67
Appendix 2: PAS (Periodic Acid Schiff) Staining Protocol 71
Appendix 3: Masson's Trichrome Staining Protocol 73
LIST OF TABLES
Table 4.1: Phytochemical composition of aqueous extracts of the three S. nigrum genotypes. 24
Table 4-2: Comparisons on intensity of staining of mucus part of the rat’s gastric glands and intensity of staining of inter-glandular connective tissue in the different treatment groups 39
Table 4-3: Protective effects of extracts from S. scabrum, S. sarrachoides and S. villosum genotypes of S. nigrum on ethanol induced gastric lesions in rats. 41
LIST OF FIGURES
Figure 2.1: A photograph of the African Nightshade (Solanum nigrum) plant 7
2.2: A schematic representation of gastric glands and the associated cell types (source: Slide share) 10
Figure 4.1: Linear necrotic lesions within the glandular portion of a rat stomach following oral administration of absolute alcohol. 26
Figure 4-2 Macrographs of stomachs of rats from the five different treatment groups. 28
Figure 4-3: Microscopic view of the normal rat stomach wall as appeared in the negative control animals. 31
Figure 4-4: Histomicrographs showing the degree of ulceration in positive control rats (i.e. those that received distilled water before ulcer induction). 33
Figure 4-5: Microscopic view of the stomach wall in negative control rats. 35
Figure 0.6: Histomicrographs showing the degree of ulceration in the five groups of rats… 37
LIST OF ABBREVIATIONS
GP Glandular portion
GU Gastric ulcer
PAS Periodic acid Schiff
NSAIDs Non-steroidal anti-inflammatory drugs
GI Gastrointestinal
CT Connective tissue
MN Mucous neck
Hcl Hydrochloric acid
Bwt Body weight
C.I Confidence interval
MeOH Methanol
DCM Dichloromethane
MT Masson’s trichrome
H & E Hematoxylin and eosin
I.p Intraperitoneal
MaUI Macroscopic ulcer index
MiUI Microscopic ulcer index
ROS Reactive oxygen species
COX Cyclooxygenase
LIST OF APPENDICES
Appendix 1: Hematoxylin and Eosin (H & E) Staining Protocol 66
Appendix 2: PAS (Periodic Acid Schiff) Staining Protocol 70
Appendix 3: Masson's Trichrome Staining Protocol 72
Appendix 4: Approval of proposal by Biosafety, Animal Use and Ethics Committee 78
CHAPTER ONE
1 INTRODUCTION
Gastric ulceration refers to lesions on the glandular part of the stomach mucosa induced by exposure to excessive hydrochloric acid and excessive pepsinogen activity (Sabiu et al., 2015). The stomach is a component of the digestive tube specialized in mechanical and chemical breakdown of food (Colville & Bassert, 2016). Like in other parts of the GIT, the wall of the stomach in mammals consists of four layers named from the inside outwards as: i) tunica mucosa comprising an epithelium, a lamina propria of loose connective tissue and a muscularis mucosae ii) tunica submucosa consisting of loose areolar connective tissue with plenty of blood vessels iii) tunica muscularis constituted by thick layers of visceral muscle fibers and iv) tunica serosa (Burkitt et al., 1993). There are four main types of epithelial cells that cover the stomach mucosal surface and extend down into the gastric pits and gastric glands. These include mucus-secreting cells, parietal/oxyntic cells which produce Hcl, peptic/chief cells which produce proteolytic enzyme pepsin and G (gastrin) cells that synthesize peptide hormone gastrin. Ulceration studies show the rat stomach to be uniquely susceptible to gastric lesions (Greaves, 2012), which in the current study were analyzed in the glandular region (part that is in constant contact with the highly corrosive gastric juice).
Gastric ulceration (GU) leads to impaired gastric function and pain and is a common cause of morbidity and mortality, with close to 53 million people developing gastric ulcers in the world each year (Vos et al., 2015). Lifestyle changes including smoking, drinking, stress and the consumption of NSADs in chronic illnesses such as arthritis are some of its predisposing factors (Moore et al., 2014; Kołodziejska & Kołodziejczyk, 2018). It is often associated with complications such as perforation, bleeding, blockage and if long standing, GU precipitates to gastric cancer (Pilotto et al., 2003, Milosavljevic et al., 2011; Bhattacharyya et al., 2014). Conventional drugs used to manage this condition include prostaglandin analogues, proton pump inhibitors, antacids and antibiotics in case of Helicobacter pylori infections. These however have toxicity as a common draw back leading to various side effects including hypersensitivity, hepatitis, hematopoietic changes and arrhythmias (Odou et al., 1999; Marcus et al., 2010). This therefore necessitates a search for anti-ulcerogenic medicines that are less toxic. Indeed, medicinal plants are highly preferred alternatives due to their easy accessibility and the fact that they are perceived to be less toxic than conventional drugs (Ernst & Hung, 2011). The use of medicinal plants to manage disease conditions dates back to human civilization (Mosihuzzaman, 2012). In developing countries, approximately four billion people (about 60% of the worldwide human population) rely on herbal products for their healthcare needs (Bodeker et al., 2005). The knowledge on indigenous plants and their medicinal uses has traditionally been passed down from one generation to the next (Miaron et al., 2004 ). Recently a lot of research has been carried out to validate commonly used folk medicines (Lahlou, 2013).
Solanum nigrum, commonly known as African night shade, is used as a vegetable and also as treatment for various ailments including gastric ulcers (Edmonds and Chweya, 1997: Ontita et al., 2016). However, scientific evidence for the use of the genotypes grown and utilized in Kenya in ulcer management is lacking. This study therefore aims at analyzing the efficacy of three S. nigrum genotypes namely: S. scabrum, S. sarrachoides and S. villosum grown in Kenya in preventing gastric ulcers.
1.1 Objectives
1.1.1 Broad objective
The overall objective of this study was to determine the anti-ulcerogenic activity of three S. nigrum genotypes (S. scabrum, S. sarrachoides and S. villosum) found in Kenya, in a rat model.
1.1.2 Specific objectives
1. To determine the phytochemical composition of the three S. nigrum genotypes grown and consumed in Kenya.
2. To describe the morphological differences in anti-ulcerogenic activity of the three S. nigrum
genotypes on the rat stomach.
3. To determine the morphometric differences in anti-ulcerogenic activity of the three S. nigrum
genotypes on the rat stomach
1.2 Null hypothesis
Solanum nigrum extracts are not anti-ulcerogenic and there are no morphological or morphometric differences in efficacy of ulcer prevention of the three Solanum nigrum genotypes.
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