ABSTRACT
Musa paradisiacae commonly known as plantain is a rhizomatous perennial crop used as a source of starchy staple for millions of people in Nigeria. Different parts of the plant have been used in the treatment of various ailments and there are claims that it has antidiarrhoeal activity. This study is therefore aimed at determining the effects of unripe Musa paradisiacae pulp and peel homogenates on castor oil-induced diarrhoea in Wistar albino rats. The qualitative phytochemical constituents of Musa paradisiacae pulp and peel were found to be flavonoids, saponins, soluble carbohydrates, tannins, reducing sugars, hydrogen cyanide, steroids, alkaloids, and glycosides. The LD50 results showed no toxicity up to 5000 mg/kg body weight. Rats were divided into 7 groups of 4 rats each. The groups were pre-treated as follows: group 1: normal saline (control); group 2: 3 mg/kg lomotil (standard drug); groups 3 and 4: 200 and 400 mg/kg unripe Musa paradisiacae pulp homogenates respectively; groups 5 and 6: 200 and 400mg/kg unripe Musa paradisiacae peel homogenates respectively; group 7: combination of unripe Musa paradisiacae pulp and peel homogenates (200/400 mg/kg respectively). After the treatments, diarrhoea was induced using castor oil. Relative to the control group 1, the treatment groups 2-7 inhibited castor oil-induced frequency of defecation and wetness of stool dose dependently but non-significantly (p>0.05). Both the pulp and peel homogenates produced non-significant decreases (p>0.05) in the distances travelled by the charcoal meal (marker) in castor oil-induced diarrhoea rats compared to the control group 1. Pre-treatment of the rats with unripe Musa paradisiacae pulp and peel homogenates decreased significantly (p<0.05) enteropooling indicated by decreases in the volume and weight of the gastro-intestinal contents relative to the control group 1. Treatment with the unripe Musa paradisiacae pulp and peel homogenates led to significant decreases (p<0.05) in the bicarbonate ion concentrations except in group 3 rats while the potassium ion concentrations increased significantly (p<0.05) in all the groups except in groups 3, 4 and 7 rats which showed non-significant decreases (p>0.05) compared to the control group 1. Sodium ion concentrations of the pre-treated groups increased non-significantly (p>0.05) except in groups 4 and 7 rats which decreased non-significantly (p>0.05) relative to the control group 1.Using everted rat intestines, the pulp and peel homogenates enhanced significant (p<0.05) influx of sodium ions into the everted sacs (serosal) and significant (p<0.05) efflux of potassium ions out of the sacs (mucosal) in relation to the control group 1. These findings reveal that unripe Musa paradisiacae pulp and peel exhibit antidiarrhoeal properties by inhibiting gastro-intestinal motility, enteropooling, wetness and frequency of defecation. They have also shown abilities to facilitate transport of electrolytes across the small intestinal membrane.
TABLE OF CONTENTS
Title Page i
Certification ii
Dedication iii
Acknowledgements iv
Abstract v
Table of Contents vi
List of Figures xii
List of Tables xiv
List of Abbreviations xv
CHAPTER ONE: INTRODUCTION – – – – – – 1
1.1 Musa paradisiacae – – – – – – – 3
1.1.1 Taxonomy of Musa paradisiacae – – – – – – 5
1.1.2 Common names of Musa paradisiacae – – – – – 5
1.1.3 Origin of Musa paradisiacae – – – – – – – 5
1.1.4 Description of Musa paradisiacae plant – – – – – 6
1.1.5 Distribution of Musa paradisiacae – – – – – – 7
1.1.6 Cultivation and storage of Musa paradisiacae – – – 8
1.1.7 Historical Uses of Musa paradisiacae – – – – – 8
1.1.8 Health benefits of Musa Paradisiacae – – – – – 8
1.1.9 Ripening process and the chemical composition of Musa paradisiacae – 9
1.2 Diarrhoea – – – – – – – – – 11
1.2.1 Definition of diarrhoea – – – – – – – 11
1.2.2 Mechanism/pathology of diarrhoea – – – – – – 12
1.2.3 Classification of diarrhoea disease – – – – – – 12
1.2.3.1 Classification based on mode of infection – – – – – 12
1.2.3.2 Classification based on duration of symptoms – – – – 12
1.2.3.3 Classification based on pathological mechanisms – – – – 13
1.2.4 Pathophysiology of diarrhoea – – – – – – 13
1.2.4.1 Intestinal inflammation and diarrhoea – – – – – 13
1.2.4.2 Oxidative damage in diarrhoea — – – – – 15
1.2.4.3 Enteric nervous system in diarrhoea – – – – – 16
1.2.4.4 Cystic fibrosis trans-membrane conductance regulator (CFTR) regulation – 17
1.2.5 Specific agents of diarrhoea – – – – – – – 17
1.2.6 Treatment of diarrhoea – – – – – – – 19
1.2.7 Potential mechanisms in the control of diarrhoea – – – – 20
1.2.8 Physiological basis of diarrhoea – – – – – 23
1.3 Phytochemicals – – – – – – – – 23
1.3.1 Terpenoids – – – – – – – – – 24
1.3.2 Alkaloids – – – – – – – – – 25
1.3.3 Phenolics – – – – – – – – – 25
1.3.4 Glycosides – – – – – – – – – 26
1.3.5 Tannins – – – – – – – – – 26
1.4 The nervous system – – – – – – – – 27
1.5 Neurotransmitters and pharmacology – – – – – – 29
1.6 Some biologically important electrolytes; sodium, potassium and bicarbonates 30
1.6.1 Hyponatremia – – – – – – – – – 30
1.6.1.1 Signs and symptoms of hyponatremia – – – – – 30
1.6.1.2 Clinical diagnosis of hyponatremia – – – – – – 31
1.6.1.3 Causes of hypernatremia – – – – – – – 31
1.6.2 Hypernatremia – – – – – – – – 31
1.6.2.1 Signs and symptoms of hypernatremia – – – – – 31
1.6.2.2 Clinical diagnosis of hypernatremia – – – – – 31
1.6.3 Hypokalemia – – – – – – – – – 30
1.6.3.1 Signs and symptoms of hypokalemia – – – – – 32
1.6.3.2 Clinical diagnosis of hypokalemia – – – – – 32
1.6.4 Hyperkalemia – – – – – – – – – 32
1.6.4.1 Signs and symptoms of hyperkalemia – – – – – 32
1.6.4.2 Clinical diagnosis of hyperkalemia – – – – – 33
1.7 Normal physiology of gut fluid and electrolyte transport – – – 31
1.8 Electrolyte transport in the jejunum and ileum – – – – 34
1.9 Castor oil – – – – – – – – 35
1.9.1 Ricin – – – – – – – – – – 34
1.9.2 Uses of castor oil – – – – – – – 36
1.10 Antidiarrhoeal drugs – – – – – – – 37
1.10.1 Nitric oxide synthase inhibitors – – – – – – 37
1.10.2 Nufenoxole – – – – – – – – – 37
1.10.3 Loperamide – – – – – – – – – 37
1.10.4 Diphenoxylate – – – – – – – – – 38
1.11 Aim of the study – – – – – – – – – 38
1.12 Specific objectives of the study – – – – – – – 39
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials – – – – – – – – – 40
2.1.2 Experimental animals – – – – – – – 40
2.1.3 Equipment – – – – – – – – – 40
2.1.4 Chemical and reagents – – – – – – – 40
2.2 Methods – – – – – – – – – 41
2.2.1 Plant collection and identification – – – – – – 41
2.2.2 Preparation of plant materials – – – – – – – 41
2.2.3 Preparation of reagents – – – – – – – – 41
2.2.3.1 Charcoal meal – – – – – – – – – 41
2.2.3.2 5% (w/v) ferric chloride solution – – – – – – 41
2.2.3.3 Ammonium solution – – – – – – – – 41
2.2.3.4 45% (v/v) ethanol – – – – – – – – 41
2.2.3.5 Aluminium chloride solution – – – – – – – 41
2.2.3.6 Dilute sulphuric acid – – – – – – – – 41
2.2.3.7 Lead acetate solution – – – – – – – – 42
2.2.3.8 Wagner’s reagent – – – – – – – – 42
2.2.3.9 Mayer’s reagent – – – – – – – – 42
2.2.3.10 Dragendorff’s reagent – – – – – – – 42
2.2.3.11 2% (v/v) hydrochloric acid – – – – – – – 42
2.2.3.12 Normal saline – – – – – – – – 42
2.2.3.13 1% (w/v) Picric acid – – – – – – – – 42
2.2.3.14 Krebs buffer – – – – – – – – – 42
2.2.3.15 0.3% glucose saline – – – – – – – – 43
2.2.3.16 Preparation of standards – – – – – – – 43
2.2.3.16.1 Potassium standard – – – – – – – – 43
2.2.3.16.2 Sodium standard – – – – – – – – 43
2.2.4 Qualitative phytochemical analysis of unripe Musa paradisiacae pulp and peel 43
2.2.4.1 Test for alkaloids – – – – – – – – 43
2.2.4.2 Test for flavonoids – – – – – – – – 43
2.2.4.3 Test for glycosides – – – – – – – – 44
2.2.4.4 Test for saponins – – – – – – – – 44
2.2.4.5 Test for tannins – – – – – – – – 44
2.2.4.6 Test for terpenoids and steroids – – – – – – 45
2.2.4.7 Test for reducing sugars – – – – – – – 45
2.2.4.8 Test for carbohydrate – – – – – – – – 45
2.2.5 Quantitative phytochemical analysis of unripe Musa paradisiacae pulp and peel 45
2.2.5.1 Quantitative determination of alkaloids – – – – – 45
2.2.5.2 Quantitative determination of flavonoids – – – – – 46
2.2.5.3 Quantitative determination of steroids – – – – – 46
2.2.5.4 Quantitative determination of cyanogenic glycosides – – – 46
2.2.5.5 Quantitative determination of tannins – – – – – – 46
2.2.6 Proximate analysis – – – – – – – – 47
2.2.6 1 Crude protein determination – – – – – – – 47
2.2.6.2 Moisture content – – – – – – – – 48
2.2.6.3 Ash – – – – – – – – – – 48
2.2.6.4 Crude fibre – – – – – – – – – 49
2.2.6.5 Crude fat – – – – – – – – – 49
2.2.7 Determination of sodium and potassium concentration – – – – 50
2.2.8 Determination of glucose – – – – – – – 50
2.2.9 Acute toxicity test of aqueous extracts of unripe Musa paradisiacae pulp and peel 51
2.2.10 Anti-diarrhoeal studies – – – – – – – 51
2.2.10.1 Castor oil-induced diarrhoea test – – – – – – 51
2.2.10.2 Castor oil-induced enteropooling test – – – – – 52
2.2.10.3 Gastro intestinal motility test – – – – – – 53
2.2.10.4 Electrolyte tests – – – – – – – 53
2.2.10.4.1 Determination of sodium ion (Na+) concentration (Teco diagnostic kit) 54
2.2.10.4.2 Determination of potassium ion (K+) concentration (Teco diagnostic kit) 55
2.2.10.4.3 Determination of bicarbonate (HCO3-) ion concentration – – – 55
2.2.10.5 Determination of the effects of the homogenates on the transport
of sodium and potassium ions across the everted rat intestine – – 56
2.2.10.5.1 Preparation of tissue – – – – – – – 56
2.2.10.5.2 Preparation of the everted sacs – – – – – – 56
2.2.10.5.3 Filling of the everted sacs – – – – – – – 57
2.3 Statistical analysis – – – – – – – – 58
CHAPTER THREE: RESULTS
3.1 The median lethal dose of unripe Musa paradisiacae
pulp and peel homogenates – – – – – – – 59
3.2 Qualitative phytochemical composition of the unripe Musa
paradisiacae pulp and peel homogenates – – – – – 61
3.3 Quantitative phytochemical composition of unripe Musa paradisiacae pulp and peel homogenates- – – – – – – – – – 63
3.4 Proximate composition of unripe Musa paradisiacae pulp and peel – – 65
3.5 Effects of unripe Musa paradisiacae pulp and peel homogenates
on castor oil-induced diarrhoea – – – – – – – 67
3.6 Effects of unripe Musa paradisiacae pulp and peel homogenates
on enteropooling in Wistar albino rats – – – – – – 69
3.6.1 Volume of the intestinal content of Wistar albino rats- – – – – 69
3.6.2 Weight of the intestinal content of Wistar albino rats- – – – – 71
3.7 Effects of unripe Musa paradisiacae pulp and peel homogenates
on gastro intestinal motility in Wistar albino rats – – – – 73
3.8 Effects of unripe Musa paradisiacae pulp and peel homogenates on the
bicarbonate levels of the small intestinal contents of Wistar albino rats 75
3.9 Effects of unripe Musa paradisiacae pulp and peel homogenates
on the potassium ion concentrations of the small intestinal contents
of Wistar albino rats – – – – – – – – 77
3.10 Effects of the of unripe Musa paradisiacae pulp and peel
Homogenates on the sodium concentrations of the small
intestinal contents of Wistar albino rats – – – – – 79
3.11 The effects of unripe Musa paradisiacae pulp and peel homogenates on the transport of sodium ions across the everted small intestinal sac – 81
3.12 The effects of unripe Musa paradisiacae pulp and peel homogenates on the transport of potassium ion across the everted small intestinal sac 83
CHAPTER FOUR: DISCUSSION
4.1 Discussion – – – – – – – – – 85
4.2 Conclusion – – – – – – – – – 88
4.3 Suggestions for Further Studies – – – – – – 89
REFERENCES – – – – – – – – – 90
APPENDICES – – – – – – – – – 106
LIST OF FIGURES
Fig. 1 Musa paradisiacae fruits – – – – – – – 3
Fig. 2 Musa paradisiacae trees – – – – – – – 4
Fig. 3 Musa paradisiacae flower. – – – – – – – 4
Fig. 4 Tubocurarine – – – – – – – – – 25
Fig. 5 Leucoanthocyanidin – – – – – – – – 26
Fig. 6 Cyanogenic glycoside (amygdalin) – – – – – – 26
Fig. 7 Organization of the nervous system – – – – – – 27
Fig. 8 Diagrammatic representation of the parasympathetic and sympathetic
nervous systems – – – – – – – – 28
Fig. 9 Hydroxidodioxidocarbonate (1−) – – – – – – 30
Fig. 10 Na+/K+-ATPase transport activity – – – – – – 33
Fig. 11 Glucose transport in intestinal epithelial cells – – – – 34
Fig. 12 Ricinoleic acid. ((9Z, 12R)-12-Hydroxyoctadec-9-enoic acid) – – 35
Fig. 13 Nufenoxole;2-[3-(5-Methyl-1,3,4-oxadiazol-2-yl)-3,3-diphenylpropyl]-2-azabicyclo[2.2.2]octane;SC-27166;2-[3-(7-azabicyclo[2.2.2]octan-7-yl)-1,1-di(phenyl)propyl]-5-methyl-1,3,4-oxadiazole – – – – 37
Fig. 14 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl] – N, N-dimethyl-2, 2-diphenylbutanamide – – – – – – – – 38
Fig. 15 1-(3-cyano-3, 3-diphenylpropyl)-4-phenylpiperidine-4-carboxylate – 39
Fig. 16 Paradoxical effect of unripe Musa paradisiacae pulp and peel
homogenates on the volume of the intestinal content of Wistar
albino rats where the pulp was inhibitory and the peel stimulatory. – – 70
Fig. 17 Inhibitory and stimulatory effects of unripe Musa paradisiacae
pulp and peel homogenates respectively on the weight of the gastro-
intestinal content of Wistar albino rats – – – – – 72
Fig. 18 Non-significant (p>0.05) inhibitory effects of unripe Musa
paradisiacae pulp and peel homogenates on gastro-intestinal
motility in Wistar albino rats – – – – – – – 74
Fig. 19 Significant (p<0.05) inhibitory and non-significant (p>0.05)
stimulatory effects of unripe Musa paradisiacae pulp and peel
homogenates on bicarbonate ion concentrations of the small
intestinal contents in Wistar albino rats – – – – – 76
Fig. 20 Dose dependent increases in the potassium ion concentrations
of the small intestinal content of Wistar albino rats treated with
unripe Musa paradisiacae pulp and peel homogenates – – – 78
Fig 21 Non-significant (p>0.05)dose dependent decreases in sodium ion
concentrations of the small intestinal contents in Wistar albino rats
treated with unripe Musa paradisiacae pulp and peel homogenates.- – 80
Fig. 22 Dose dependent increases in the influx of sodium ions
into the everted sacs (serosal) in the presence of unripe
Musa paradisiacae pulp and peel homogenates. – – – – 82
Fig. 23 Dose dependent increases in the efflux of potassium ions
outside the everted rat intestine in the presence of unripe Musa
paradisiacae pulp and peel homogenates – – – – – 84
LIST OF TABLES
Table 1: Nutritional value of Musa paradisiacae pulp per 100g (3.5oz) – 11
Table 2: Functions of the autonomic nervous system – – – – 28
Table 3: The median lethal dose of unripe Musa paradisiacae
pulp and peel homogenates – – – – – – – 60
Table 4: Qualitative phytochemical composition of unripe Musa
paradisiacae pulp and peel homogenates- – – – – – 62
Table 5: Quantitative phytochemical composition of unripe
Musa paradisiacae pulp and peel – – – – – – 64
Table 6: Proximate composition of unripe Musa paradisiacae pulp and peel – 66
Table 7: Inhibition of castor oil-induced frequency of defecation of unripe
Musa paradisiacae pulp and peel homogenates in Wistar albino rats – 68
LIST OF ABBREVIATIONS
WHO- – -WORLD HEALTH ORGANISATION
CAMP – – -CYCLIC ADENOSINE MONOPHOSPHATE
CGMP – –CYCLIC GUANOSINE MONOPHOSPHATE
GIT – –GASTRO INTESTINAL TRACT
ENS- – -ENTERIC NERVOUS SYSTEM
CFTR – — CYSTIC FIBROSIS TRANS-MEMBRANE CONDUCTANCE REGULATOR
EHEC….ENTEROHAEMORRHAGIC E.COLI
ETEC….ENTEROTOXIGENIC E COLI
EPEC….ENTEROPATHOGENIC E. COLI
EIEC…ENTERO INVASIVE E. COLI
DAEC- – -DIFFUSIVELY ADHERENT E. COLI
EAEC- – -ENTEROAGGREGATIVE E.COLI
SETs – – -STABLE STAPHYLOCOCCAL ENTEROTOXINS
TSST-1 – – -TOXIC SHOCH SYNDROME TOXIN
HIV- – -HUMAN IMMUNO DEFICIENCY VIRUS
ROS – – -REACTIVE OXYGEN SPECIES
RNS – – -REACTIVE NITROGEN SPECIES
NSAIDs – – -NON STEROIDAL ANTI-INFLAMMATORY DRUGS
COX- – -CYCLO-OXYGENASE
LOX – – -LIPO-OXYGENASE
NO – – -NITRIC OXIDE
ORT – – -ORAL REHYDRATION THERAPY
SGLT – – -SODIUM-GLUCOSE TRANSPORTER
ANS – – -AUTONOMIC NERVOUS SYSTEM
SNS- – -SYMPATHETIC NERVOUS SYSTEM
PSNS – – -PARASYMPATHETIC NERVOUS SYSTEM
ATP – – -ADENOSINE TRIPHOSPHATE
ECG – – -ELECTROCARDIOGRAM
DRA – – -DOWN REGULATED IN ADENOMA
CLD – – -CHLORIDE DIARRHOEA
FDA – – -FOOD AND DRUG ADMINISTRATION
Na+ / K+ ATPASE – – -SODIUM/POTASSIUM ADENOSINE TRIPHOSPHATASE
SCFAs – – – SHORT CHAIN FATTY ACIDS
CHAPTER ONE
INTRODUCTION
The use of traditional medicines in West Africa is probably as old as the duration of human settlement in the region (Abdul-aguye, 1997). A medicinal plant provides an important source of new chemical substances with potential therapeutic effects. These have been used in traditional medicine for the treatment of several diseases and aliments (Mukerjee et al., 1998). It is already important to the global economy with demand steadily increasing not only in developing countries but also in industrialized countries (Sofowara, 1993).
Herbalism or herbal medicine is the use of plants for medicinal purposes, and the study of such use (Briskin, 2000). Herbal medicine is still the mainstay of about 75 – 80% of the world population, mainly in the developing countries, for primary health care (Kamboj, 2000). Plants have been the basis for medical treatments through much of human history, and such traditional medicine is still widely practiced today (Briskin, 2000). This is primarily because of the general belief that herbal drugs are without any side effects besides being cheap and locally available (Gupta and Raina, 1998). Modern medicine recognizes herbalism as a form of alternative medicine as the practice of herbalism is not strictly based on evidence gathered using the scientific method (Talalay, 2001). According to the World Health Organization (WHO), the use of herbal remedies throughout the world exceeds that of the conventional drugs by two to three times (Evans, 1994). The use of plants for healing purposes predates human history and forms the origin of much modern medicine. Modern medicine, does, however, make use of many plant-derived compounds as the basis for evidence-tested pharmaceutical drugs, and phytotherapy works to apply modern standards of effectiveness testing to herbs and medicines that are derived from natural sources (Talalay, 2001). Examples include aspirin (willow bark), digoxin (from foxglove), quinine (from cinchona bark), and morphine (from the opium poppy) (Vickers and Zollman, 1999). Currently, a number of medicinal plants with antidiarrhoeal and antimicrobial properties are used in traditional herbal practice in many countries of the world. So it is important to identify and evaluate commonly available natural drugs that could be used against any type of diarrhoeal disease.
A number of herbs are thought to likely have adverse effects (Talay, 2001). Furthermore, “adulteration, inappropriate formulation, or lack of understanding of plant and drug interactions have led to adverse reactions that are sometimes life threatening or lethal (Elvin-Lewis, 2001). Proper double-blind clinical trials are needed to determine the safety and efficacy of each plant before they can be recommended for medical use (Vickers, 2007). Although many consumers believe that herbal medicines are safe because they are “natural”, herbal medicines and synthetic drugs may interact, causing toxicity to the patient. Herbal remedies can also be dangerously contaminated, and herbal medicines without established efficacy, may unknowingly be used to replace medicines that do have corroborated efficacy (Ernst, 2007). The World Health Organization (WHO), the specialized agency of the United Nations (UN) that is concerned with international public health, published quality control methods for medicinal plant materials in 1998 in order to support WHO Member States in establishing quality standards and specifications for herbal materials, within the overall context of quality assurance and control of herbal medicines (WHO, 2010).
There are different methods of herbal preparations and the exact composition of an herbal product is influenced by the method of extraction. They are:
1) Tisanes or herbal teas; are the resultant liquid of extracting herbs into water (Green, 2000). The methods used are, infusions (hot water extracts of herbs), decoctions (long term boiled extracts usually of harder substances like roots and bark) and maceration (old infusion of plants with high mucilage content) (Green, 2000).
2) Tinctures; alcoholic extracts of herbs generally stronger than tisanes (Green, 2000).
3) Syrups; extracts of herbs made with syrups or honey (Green, 2000).
In developing countries, diarrhoea continues to be one of the leading causes of mortality and morbidity in children less than 5 years old. According to World Health Report, diarrhoea is the cause of 3.3% of all deaths. Worldwide distribution of diarrhoea accounts for more than 5-8 million deaths each year in children. The incidence of diarrhoeal disease still remains high despite the effort by many government and international organizations to reduce it. Nigeria, the fourth largest economy in Africa with an estimated per capita income of $350 has over half of its population living in poverty (WHO, 2007). This implies that very few people can afford orthodox medicine in curing diseases. Use of traditional medicines to combat the consequences of diarrhoea has been emphasized by WHO in its Diarrhoea Control Programme. It is therefore important to identify and evaluate available natural drugs as alternatives to current antidiarrhoeal drugs, which are not always free from adverse effects. Several studies have shown the beneficial effects of a number of medicinal
