Pharmacological Evaluation and Characterization of the Antiulcer Constituents of Stem Bark Extract of Bridelia ferruginea Benth (Euphorbiaceae).
ABSTRACT
Peptic ulcer disease (PUD) is a sore in the lining of the stomach or duodenal mucosa. The search for an ideal antiulcer drug continues and has also been extended to medicinal plants. Bridelia ferruginea Benth (Euphorbiaceae) is a plant used in traditional medical practice in South West Nigeria.
This study was aimed at evaluating the antiulcer activity and mechanisms of the extract of the stem bark and to isolate the bioactive constituents responsible for the antiulcer activity.
Methanol extract (ME) was obtained by cold maceration and concentration in vacuo. ME was partitioned in chloroform-methanol-water (2:2:1) mixture to obtain the chloroform (CF) and aqueous methanol (AMF) fractions.
The extract and fractions were subjected to biological activity-guided screening using indomethacin-induced ulcer as activity-guide.
Based on higher ulcer protection given by CF, it was fractionated in a silica gel and eluted with gradient mixtures of n-hexane-ethyl acetate to obtain six broad fractions (I – VI). Fractions III and VI offered the highest protection on screening for biological activity.
Purification of fractions III and VI in a sephadex LH-20 column with methanol as eluent gave compounds I (BF1) and II (BF2) respectively.
The antiulcer activity of BF1 and BF2 was done using the activity-guide and the structural identities established using nuclear magnetic resonance (1H-NMR, 13C-NMR) and electron impact mass (EIM) spectroscopies. The extract was subjected to phytochemical analysis using conventional methods.
The oral acute toxicity of ME was determined in mice. The antiulcer activity of ME, bioactive column fractions and isolated compounds was investigated using indomethacin, ethanol, cold-restraint stress and pyloric ligation-induced ulcers in rats.
The mechanisms of antiulcer activity were studied using gastric acid secretion induced by pyloric ligation in rats, proton pump inhibition using inhibition of H+ K+ ATPase activity in vitro.
Determination of the roles of endogenous nitric oxide and sulfhydryl compounds using the effects of L-NAME (L-nitroarginine methylester) and NEM (N-ethylmaleimide) respectively on ulcer indices in ethanol-induced ulcer and antioxidant activity using DPPH radical scavenging activity.
The results showed that ME tested positive to saponins, reducing sugars, tannins, carbohydrates, flavonoids, glycosides, alkaloids, steroids, proteins and terpenoids. No lethality was observed in the mice on oral administration of doses up to 5000 mg/kg.
There were no obvious signs of abnormal behavioural changes in the mice. The extract, fractions and isolated compounds produced significant (p < 0.05) dose-related inhibition of indomethacin, ethanol, cold restraint stress and pyloric ligation-induced ulcers.
The isolated compounds, BF1 and BF2, significantly (p < 0.05) decreased the total acid and volume of gastric secretion and elevated the pH. The fractions and isolated compounds significantly (p < 0.05) inhibited the activity of H+ K+ ATPase in a dose-dependent manner.
The isolated compounds, BF1 and BF2 did not increase gastric lesion indices in L-NAME pre-treated rats but they significantly (p < 0.05) increased ulcer indices in the NEM pre-treated rats. The extract, fractions and isolated compounds scavenged DPPH radical in a dose-dependent manner.
Comparison of the spectral data of BF1 and BF2 with the published libraries of isolated compounds revealed their identities to be β-sitosterol and β- sitosterol-3-O-βD-glucopyranoside respectively.
TABLE OF CONTENTS
Title Page……. iii
Certification…. iv
Dedication…… v
Acknowledgement…. vi
Table of Contents……….. vii
List of Abbreviations……. xii
List of Figures… xvi
List of Tables……. xviii
Abstract…. xx
CHAPTER ONE: INTRODUCTION
- Introduction…… 1
- Epidemiology…. 2
- Signs/symptoms….. 3
- Diagnosis of peptic ulcer disease…. 5
- Complications of peptic ulcer disease…….. 5
- Laboratory tests…… 5
- Visualization of ulcers……….. 6
- Pathophysiology of PUD. 7
- Risk factors for PUD…….. 8
- Acid secretion…… 8
- Helicobacter pylori…. 9
- NSAIDs…… 11
- Free radicals….. 13
- Life style risk factors…. 16
- Smoking……. 16
- Alcohol…… 16
- Stress……… 17
- Endogenous gastroprotective mechanisms….. 19
- Physiological gastric mucosal barrier………. 19
- Mucus-bicarbonate-phospholipid barrier…. 19
- Epithelial cells………. 20
- Mucosal cell renewal….. 21
- Mucosal blood flow…………. 22
- Sensory innervation……. 23
- Prostaglandins……….. 24
- Neurohormonal mechanism…….. 24
- Mucosal immune system………. 26
- Endogenous gastroprotective mediators……… 26
- Nitric oxide…. 26
- Heat shock protein.. 27
- Prostaglandins…….. 28
- Lipoxins……. 29
- Mucus…….. 30
- Sulfhydryl group…. 30
- Hydrogen sulfide……… 30
- Hemeoxygenase-1-enzyme….. 31
- Neuropeptides…… 32
- Peroxisome proliferation-activated receptor…… 33
- Growth factors…. 34
- Proteinase-activated receptors……… 35
- Therapeutic interventions….. 35
- Goals of therapy….. 36
- Conventional antiulcer drugs…….. 36
- Non-specific therapeutic measures. 42
- Herbal and natural products for the treatment of PUD…. 42
- Non-conventional gastroprotective drugs……. 52
- Plant botanical profile……… 54
1.13.2 Plant description……… 54
- Geographical distribution…… 58
- Ethnomedicinal uses…….. 58
- Literature review…… 59
- Aims of the study…………… 60
CHAPTER TWO: MATERIALS AND METHODS
- Materials……………. 61
- Animals……… 61
- Chemicals, solvents and reagents…… 61
- Drugs……….. 61
- Methods……….. 61
- Collection of plant materials….. 61
- Extraction……………….. 62
- Phytochemical analysis of extract………….. 62
2.2.4 Fractionation of extract……………………. 64
- Biological activity guided studies …………… 65
- Characterization of isolated compounds……… 65
- Pharmacological tests………. 67
- Acute toxicity tests……………. 67
- Antiulcer activity tests……. 67
- Indomethacin-induced ulcer test……. 67
- Absolute ethanol-induced ulcer test….. 69
- Cold restraint stress-induced ulcer test… 70
- Pyloric ligation-induced ulcer test………….. 70
- Antiulcer mechanisms tests…………. 71
- Gastric acid secretion tests……… 71
- H+ K+ ATPase inhibition activity test………. 72
- Role of endogenous nitric oxide and sulfhydryl compounds…… 73
- Antioxidant (DPPH) radical scavenging activity test…………. 75
- Statistical analysis…… 76
CHAPTER THREE: RESULTS
- Extraction and fractionation of extract…. 77
- Phytochemical constituents of extract…. 77
- Acute toxicity and lethality of extract….. 77
- Physical characteristics of isolated compounds……….. 77
- Spectral data and structures of isolated compounds………… 77
- Effect of extract, fraction and isolated compounds on indomethacin-induced ulcer……… 86
- Effect of extract, fraction and isolated compounds on absolute ethanol-induced ulcer….. 86
- Effect of extract, fraction and isolated compounds on cold restraint stress-induced ulcer93
- Effect of isolated compounds on pyloric ligation-induced ulcers……. 93
- Effect of isolated compounds on gastric acid secretion…………….. 93
- Effect of isolated compounds on H+ K+ ATPase inhibition activity…………………………….. 98
- Effect of isolated compounds on gastric lesion in L-NAME pre-treated rats………………… 98
- Effect of isolated compounds on gastric lesion in NEM pre-treated rats……………………… 98
- Effect of extract, fractions and isolated compounds on DPPH activity……………………….. 98
CHAPTER FOUR: DISCUSSION AND CONCLUSION
- Discussion…. 105
- Conclusion……. 114
REFERENCES…. 115
APPENDIX
INTRODUCTION
1.1 Peptic Ulcer Disease
Peptic ulcer is a sore that forms in the lining of the stomach or the duodenum. It encompasses gastric and duodenal ulcers and is the most prevalent gastrointestinal disorder (Prabha et al., 2011).
Clinically, it presents as abdominal stress most often in the upper quandrant of the abdomen and epigastric region (Mayty, 2003; Ezekwesili et al., 2014). Peptic ulcer disease (PUD) is a chronic disease which impairs quality of life with increased morbidity and mortality (Shobha and Jamadar, 2013).
It is associated with necrosis, infiltration of neutrophils, reduction in blood flow, and induction of oxidative stress as well as secretion of inflammatory mediators (De Souza et al., 2011; Viana et al., 2013).
Ulceration refers to a process of inflammation in which an epithelial surface of the skin, gastric epithelium, colonic mucosa, and bladder epithelium has become necrotic and eroded, often associated with subepithelial acute and chronic inflammation (Tripathi, 2008; Yadav et al., 2013).
PUD may manifest as superficial, deep or perforated erosions equal to or greater than 0.5 cm of the mucosal lining of the stomach, pyloric channels, duodenum and at or near the site of surgical anastomosis (post-operative ulcers) (Dhasan et al., 2010; Kalra et al., 2011).
The major types of peptic ulcer disease are gastric and duodenal ulcers. About 98% of peptic ulcers are either in the first portion of the duodenum or in the stomach in a ratio of about 4:1 (James and Vinay, 2003).
REFERENCES
Abdiev, S., Ahn, K. S., Khadjibaev, A., Malikov, Y., Bahramov, S., Rakhimov, B., Sakamoto, J., Kodera, Y., Nakao, A. and Hamajima, N. (2010): Helicobacter pylori infection and cytokine gene polymorphisms in Uzbeks. Nagoya J. Med. Sci. 72: 167-172.
Abdulla, M.A., Ali, H.M., Ahmed, K.A., Noor, S.M. and Ismail, S. (2009): Evaluation of the anti-ulcer activities of Morus alba extracts in experimentally-induced gastric ulcer in rats. Biomed. Res. 20 (1): 35–39.
Abdulla, M.A., Ahmed, K.A., Bayaty, F.H. and Masood, Y. (2010): Gastroprotective effect of Phyllanthus niruri leaf extract against ethanol-induced gastric mucosal injury in rats. Afri. J. Pharm. Pharmacol. 4 (5):226-30.
Addae-Mensah, I. and Achenbach, H. (1985): Terpenoids and flavonoids of Bridelia ferruginea. Phytochem. 24 (8): 1817-1819.
Addae-Mensah, I. (1992): Towards a rational scientific basis for herbal medicine: A phytochemist’s two decade contribution. Accra: Ghana University Press.
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