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ANTIULCEROGENIC AND IMMUNOSTIMULATORY PROPERTIES OF THE AQUEOUS EXTRACT OF THE LEAVES OF FICUS CAPENSIS IN WISTAR ALBINO RATS
ABSTRACT
Ficuscapensis leaves have always played a great role in preventing, controlling and alleviating various disease conditions. This study was aimed at validating the traditional use of the leaves of Ficuscapensisin folk medicine as an anti- ulcer and immunostimulatory agent. Diclofenac sodium (100mg/kg) body weight was administered intraperitoneally to induce ulcer. Seven groups(five rats each)were used. Groups 2(150mg ranithidine),4,5,6and7 served as the ulcer test groups while group1 and 3 served as ulcer untreated group(negative and positive controls respectively).After 24 hour fasting, all the rats were sacrificed and the stomach was removed for observation of ulcer score, ulcer index and ulcer indices(SOD,CAT, MDA and GSH).Five groups(five rats each) were used to investigate delayed type hypersensitivity(DTH),humoral antibody(HA)and Myelosuppression. Groups 2,3, 4 and 5 (Levamisol) served as the test groups while group1 was the negative control.DTH in rats was sensitized by subcutaneous (SC) injection of 0.1ml of 109 cells /ml sheep red blood cells (SRBCs) (day 0) in the planter region of the right hind foot paw and was challenged on day 5 by SC injection of the same concentration of antigen into the left hind paw. The oedema produced by antigenic challenge in the left hind paw was measured with a micrometer screw guage. HA in rats was immunized by ip injection of 0.2ml of 109SRBCs/ml on day 0 and was challenged by injecting the same concentration on day 7.Primary antibody titre was determined on day 7(before the challenge) and the secondary titre was determined on day 14 by the haemagglutination technique. The highest dilution showing visible haemagglutination was taken as the antibody titre. Cyclophosphamide (30 mg/kg, i.p.) was used to induce myelo suppression and levamisole (50 mg/kg)was used as standard immune stimulating agents. After five days, blood sample was collected using rectobulba plexus in the eye. The before induction, after induction and after treatment blood samples obtained were used to determine the haematological parameters(WBC,Hb, PCV, RBC).The qualitative and quantitative phytochemical analysis showed that the aqueous extract contained: reducing sugar(543.47 ± 0.004), saponins (2.39 ± 0.0032), tannin(7.57± 0.0015), flavonoid (1.27 ± 0.0321), soluble carbohydrates(3.92 ± 0.0030), alkaloid(3.34 ± 0.0015), steroid(1.04 ± 0.0321), hydrogen cyanide(1.61 ± 0.0026), glycoside(2.37 ± 0.0025), terpenoid (0.89 ± 0.0025),fats and oil.The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. The ulcer index decreased significantly (p<0.05) in the treatment groups compared to positive control (group 3).MDA activity decreases significantly(p<0.05) compared to group 3. The antioxidant enzymes: CAT,GSH and SOD activities of the test groups significantly increased (p <0.05) compared to group 3.DTH SRBC challenged rats showed a comparable increase in thickness of rat footpad 24hr and 48hr after challenge. Administration of extract produced significant increase (p<0.05) in footpad swelling of the rats compared to control. Significant increase (p<0.05) in the primary and secondary SRBCs – specific mean antibody titre values after treatment was observed compared to control. Cyclophosphamide caused a significant (p<0.05) decrease in haematological parameters. Treatment with extract or levamisol resulted in a restoration of the bone marrow activity. Haematological parameters of the test groups significantly increased (p <0.05) compared to control. The findings indicated that the extract could be used in the management of ulcer and immunosuppressive disorders.
TABLE OF CONTENTS PAGE
Title Page – – – – – – – – – – i
Certification – – – – – – – – – – ii
Dedication – – – – – – – – – – iii
Acknowledgement – – – – – – – – – iv
Abstract – – – – – – – – – – v
Table of Content – – – – – – – – – vi
List of Figures – – – – – – – – – – xiii
List of Tables – – – – – – – – – – xv
CHAPTER ONE: INTRODUCTION
1.1 Definition of ulcer – – – – – – – – 1
1.1.1 Brief history of peptic ulcer diseases – – – – – 1
1.1.2 Pathogenesis of peptic ulcer – – – – – – 2
1.2 Factors that produce ulceration – – – – – – 3
1.2.1 Endogenous factors producing ulceration – – – – – 3
1.2.1.1 Acetylcholine – – – – – – – – 3
1.2.1.2 Gastrin – – – – – – – – – 4
1.2.1.3 Histamine – – – – – – – – – 5
1.2.1.4 Somatostatin and Cholecystokinin – – – – – – 6
1.2.1.5 Calcium ion as a second messenger – – – – – 6
1.2.1.6 Genetic factors – – – – – – – – 6
1.2.2 Exogenous factors producing ulceration – – – – – 6
1.2.2.1 Helicobacter pyroli – – – – – – – – 6
1.2.2.2 Non-steroidal anti-inflammatory drugs – – – – – 8
1.2.2.3 Ethanol – – – – – – – – – 10
1.2.2.4 Cigarette smoking – – – – – – – – 10
1.2.2.5 Diet – – – – – – – – – – 10
1.2.2.6 Psychological factors (Stress ulcers) – – – – – 11
1.2.3 Free radicals/ reactive oxygen species – – – – – 12
1.3 Diagnosis of ulcer – – – – – – – – 14
1.3.1 Endoscopy and Radiology approaches to the diagnosis of ulcer – – 14
1.3.1.1 Culture – – – – – – – – 14
1.3.1.2 Histological assessment – – – – – – – 15
1.3.1.3 Rapid urease tests – – – – – – – – 15
1.3.1.4 Polymerase chain reaction – – – – – – – 15
1.3.2 Non-endoscopic diagnosis of ulcer – – – – – 15
1.3.2.1 Antibody test – – – – – – – – 15
1.3.2.2 Urea breath test – – – – – – – – 16
1.3.2.3 Stool antigen test – – – – – – – – 16
1.4 Therapy for peptic ulcer – – – – – – – 16
1.4.1 Non-pharmacological treatment of peptic ulcer disease – – – 16
1.4.2 Pharmacological Treatment of peptic ulcer disease – – – 17
1.5 An overview of the immune system – – – – – 18
1.5.1 Immunomodulation – – – – – – – 18
1.5.1.1 Immunostimulation – – – – – – – 18
1.5.1.2 Immunosuppression – – – – – – – 19
1.6 Humoral immunity – – – – – – – – 20
1.7 Cell-mediated immunity – – – – – – – 21
1.8 Anaemia – – – – – – – – – 22
1.8.1 Types of anaemia – – – – – – – – 23
1.8.1.1 Iron deficiency anaemia – – – – – – – 23
1.8.1.2 Haemolyticanaemia – – – – – – – 23
1.8.1.3 Acute haemorrhagicanaemia – – – – – – 23
1.8.1.4 Chronic haemorrhagic anemia – – – – – – 24
1.8.1.5 Perniciousanaemia – – – – – – – – 24
1.8.1.6 Aplastic anaemia – – – – – – – – 24
1.9 Haematological indices – – – – – – – 25
1.9.1 Red blood cells (Erythrocytes) – – – – – – 25
1.9.2 Packed Cell Volume (PCV) – – – – – – 25
1.9.3 Haemoglobin – – – – – – – – 25
1.9.4 Total white blood cells (WBCs) count – – – – – 26
1.10 Haematopoiesis – – – – – – – – 26
1.10.1 Vitamin B12 – – – – – – – – – 27
1.10.2 Folic acid – – – – – – – – – 27
1.10.3 Vitamin C (Ascorbic Acid) – – – – – – 28
1.10.4 Iron – – – – – – – – – – 28
1.10.5 Erythropoietin (EPO) – – – – – – – 29
1.11 Phytochemicals – – – – – – – – 30
1.11.1 Phytochemical constituents in plants – – – – – 30
1.11.1.1 Alkaloids – – – – – – – – – 31
1.11.1.2 Flavonoids – – – – – – – – – 31
1.11.1.3 Tannins – – – – – – – – – 32
1.11.1.4 Steroids – – – – – – – – – 33
1.11.1.5 Terpenoids – – – – – – – – – 33
1.11.1.6 Saponins – – – – – – – – – 34
1.12 Medicinal plants – – – – – – – – 34
1.12.1 Ficus capensis – – – – – – – – 35
1.12.1.1 Botanical outline of Ficus capensis – – – – – 35
1.12.1.2 Uses of Ficus capensis – – – – – – – 35
1.12.1.3 Taxonomy of Ficus capensis – – – – – – 37
1.13 Aim and Objectives of the study – – – – – – 37
1.13.1 Aim of the study – – – – – – – – 37
1.13.2 Specific objectives of the study – – – – – – 37
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials – – – – – – – – – 38
2.1.1 Plant material – – – – – – – – 38
2.1.2 Animal – – – – – – – – 38
2.1.3 Chemicals and Reagents – – – – – – – 38
2.1.3.1 Chemicals – – – – – – – – – 38
2.1.3.2 Reagents – – – – – – – – – 39
2.2 Methods – – – – – – – – – 41
2.2.1 Aqueous extraction – – – – – – – – 41
2.2.2 Determination of extract yield – – – – – 41
2.2.3 Phytochemical analysis – – – – – – – 41
2.2.3.1 Test for Terpenoids – – – – – – – 41
2.2.3.2 Test for Glycoside – – – – – – – – 41
2.2.3.3 Test for Tanin – – – – – – – 41
2.2.3.4 Test for Cyanide – – – – – – – – 42
2.2.3.5 Test for Soluble carbohydrate – – – – – – 42
2.2.3.6 Test for Steroid – – – – – – – – 42
2.2.3.7 Test for Saponin – – – – – – – – 42
2.2.3.8 Test for Flavonoid – – – – – – – – 42
2.2.3.9 Test for Reducing sugar – – – – – – – 42
2.2.3.10 Test for Alkaloid – – – – – – – – 43
2.2.3.11 Test for Oil – – – – – – – – – 43
2.2.4 Acute toxicity and lethality – – – – – – 43
2.2.5 Induction of ulcer – – – – – – – – 43
2.2.5.1 Experimental design of ulcer – – – – – – 44
2.2.5.2 Ulcer index – – – – – – – – – 44
2.2.6 Determination of lipid peroxidation (Malondialdehyde) concentration – 45
2.2.7 Assay of catalase activity – – – – – – – 45
2.2.8 Assay of superoxide dismutase (SOD) activity – – – – 46
2.2.9 Determination of glutathione (GSH) concentration – – – 47
2.2.10 Immunomodulatory methods – – – – – 48
2.2.10.1 Determination of delayed- Type Hypersensitivity (DTH) reaction – 48
2.2.10.2 Determination of Antibody titration (Humoral mediated response) – 48
2.2.10.3 Determination of cyclophosphamide induced myelosuppression in rats – 49
2.2.10.3.1Experimental Design of cyclophosphamide induced myelosuppression in rats 49
2.2.10.3.2Determination of packed cell volume – – – – – 50
2.2.10.3.3Determination of haemoglobin (Hb) concentration – – – 50
2.2.10.3.4Determination of Red Blood Cells counts (RBCs) – – – – 51
2.2.10.3.5Determination of Total White Blood Cell count – – – – 52
2.3 Statistical Analysis – – – – – – – – 53
CHAPTER THREE: RESULTS
3.1 Percentage yield of Aqueous Leaf Extract of Ficuscapensis – 54
3.2 Phytochemical Analysis – – – – – – – 55
3.2.1 Qualitative Phytochemical Screening of Ficuscapensis – – – 55
3.2.2 Quantitative Phytochemical Constituents of Aqueous Leaf Extract
of F. capensis – – – – – – – – – 57
3.3 Biological Effects of F. capensis – – – – – – 59
3.3.1 Acute Toxicity (LD50) test of aqueous extract of the leaves of F. capensis 59
3.3.2 Ulcerogenic Activity of the Extract – – – – – 61
3.3.2.1 Ulcer Index and Preventive Ratio of aqueous extract of the leaves of F. Capensis – – – – – – – – – 61
3.3.3 Enzymatic antioxidant – – – – – – – 63
3.3.3.1 Effect of aqueous extract of Ficus capensis leaves on Mean Catalase Activity on Diclophenac sodium-induced Gastric Lesion in
Wistar Albino Rats — – – – – – – – 63
3.3.3.2 Effect of aqueous extract of Ficus capensis leaves on Mean Malondialdehyde
(MDA) concentration on Diclophenac sodium-induced Gastric Lesion in Wistar Albino Rats – – – – – – – – 65
3.3.3.3 Effect of aqueous extract of Ficus capensis leaves on Mean superoxide dismutase (SOD) Activity on Diclophenac sodium-induced Gastric Lesion in Wistar Albino Rats – – – – – – 67
3.3.3.4 Effect of aqueous extract of Ficus capensis leaves on Mean Glutathione (GSH)
Activity on Diclophenac sodium-induced Gastric Lesion in Wistar Albino Rats – – – – – – – – – 69
3.4 Immunomodulatory Activities of the Extract – – – – 71
3.4.1 Effect of the extract on Delayed – Type Hypersensitivity (DTH) reaction – 71
3.4.2 Effect of the extract on Humoral antibody synthesis – – – 73
3.4.3 Cyclophosphamide Induced myelosuppression – – – – 75
3.4.3.1 Treatment with Cyclophosphamide – – – – – 75
3.4.3.2 Treatment with levamisol – – – – – – – 75
3.4.3.3 Effects of aqueous extract on cyclophosphamide induced myelosuppression
on the Hb concentration of rats before and after induction with treatments 75
3.4.3.4 Effects of aqueous extract on cyclophosphamide induced myelosuppression
on the RBC of rats before and after induction with treatments – – 77
3.4.3.5 Effects of aqueous extract on cyclophosphamide induced myelosuppression
on the PCV of rats before and after induction with treatments – – 79
3.4.3.6 Effects of aqueous extract on cyclophosphamide induced myelosuppression
on the WBC of rats before and after induction with treatments – – 81
CHAPTER FOUR: DISCUSSION
4.1 Conclusion – – – – – – – – – 92
4.2 Suggestions for Further studies – – – – – – 92
REFERENCES – – – – – – – – – 93
APPENDICES – – – – – – – – – 109
LIST OF FIGURES
Fig. 1: Role of aggressive and defensive factors in the pathogenesis of peptic ulcer
(Adopted from Robbin and Cotran’s Pathologic Basis of Disease) – – 3
Fig. 2: The role of gastrin in the regulation of acid secretion – – – – 4
Fig. 3: Mechanism involved in regulation of gastric acid secretion by the parietal cells – 5
Fig.4: Structure of Diclofenac-sodium – – – – – – – 10
Fig. 5: Pathway showing several mechanisms of free radical generation in the body – 12
Fig. 6: Classification of various drugs used in the treatment of peptic ulcer 17
Fig 7: Cyclophosphamide – – – – – – – – – 20
Fig. 8: Ficuscapensis – – – – – – – – – 36
Fig. 9: Effect of aqueous leave extract of Ficuscapensis on Mean Catalase Activity
on Diclophenac sodium-induced Gastric Lesion in Wistar Albino Rats – – 64
Fig. 10:Effect of aqueous leave extract of Ficuscapensis on mean malondialdehyde (MDA) concentration on Diclophenac sodium-induced Gastric Lesion in Wistar Albino Rats – – – – – – – – – 66
Fig.11: Effect of aqueous leave extract of Ficuscapensison mean superoxide dismutase (SOD) Activity on Diclophenac sodium-induced Gastric Lesion in
Wistar Albino Rats – – – – – – – – 68
Fig.12: Effect of aqueous leave extract of Ficuscapensis on mean Glutathione (GSH) Activity on Diclophenac sodium-induced Gastric Lesion in Wistar Albino Rats – – – – – – – – – 70
Fig. 13:Delayed – Type Hypersensitivity (DTH) reaction – – – 72
Fig. 14:Humoral Antibody Synthesis – – – – – – 74
Fig.15: Effect of aqueous extract on cyclophosphamine induced myelosuppression on Hb of rats before and after induction with treatments – – – – 76
Fig.16: Effect of aqueous extract on cyclophosphamide induced myelosuppression on RBC of rats before and after induction – – 78
Fig. 17:Effect of aqueous extract of cyclophosphamide induced myelosuppression on PCV rats before and after induction with treatments – – – 80
Fig.18: Effect of aqueous extract on cyclophosphamide induced myelosuppression on WBC of rats before and after induction with treatments – – – 82
LIST OF TABLES
Table 1: Preliminary Phytochemical Screening Aqueous Leaf Extract of Ficuscapensis – 56
Table 2: Quantitative Phytochemical Constituents of Aqueous Leaf Extract of Ficuscapensis-58
Table 3: The median lethal dose of aqueous extract of the leaves of F. Capensis – – 60
Table 4: Effect of aqueous extract of the leaves of F. Capensis on Diclophenac sodium -induced gastric lesion on Wistar albino rats – – – – 62
CHAPTER ONE
INTRODUCTION
Plant materials are sources of shelter, food and medicinal compounds which have been known to play a dominant role in the maintenance of human health in most rural communities in the developing countries (Oduola et al., 2005). Herbal medicine is fast emerging as an alternative treatment to available synthetic drugs for the treatment of diseases possibly due to lower cost, availability, fewer adverse effects and perceived effectiveness (Ubaka et al., 2010). The World Health Organisation (WHO) has shown great interest in plant derived medicines which have been described in the folklore medicines of many countries (Mukheerjee, 2002). However, the historic role of medicinal plants in the treatment and prevention of diseases and their role as catalysts in the development of pharmacology do not however, assure their safety for uncontrolled use by an uninformed public (Matthews et al., 1999). In Enugu State of Nigeria, many plants are used in herbal medicine to treat diseases, as well as to promote rapid healing of wound and sores (Dimo et al., 2002).
1.1 Ulcer
An inflammatory, usually suppurating, lesion on the skin or internal mucous surface that results in necrosis is referred to as ulcer (Nwodo, 2012). A peptic ulcer also known as ulcer pepticum or peptic ulcer disease (PUD) is defined as a disruption of the mucosal integrity of stomach, duodenum or oesophagus leading to a local defect or excavation due to active inflammation (Adreoli et al., 2008).
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