ABSTRACT
This study investigated the anti-diarrhoeal potential of Gouania longipetala crude extract and its six different fractions in different anti-diarrhoeal study groups in rats, to establish a scientific basis for its use in traditional medicine as an anti-diarrhoeal. The crude ethanol extract was subjected to phytochemical, gas chromatography-mass spectrometry (GC-MS), molecular docking and FTIR analyses. Acute toxicity evaluation of the crude extract was also done to establish its LD50 value. Its crude extract was evaluated using different doses (200, 400, 600 and 800mg/kg body weight) orally for antidiarrheal activity using castor oil-induced diarrhea, charcoal meal transit time and castor oil-induced enteropooling in different groups of albino rats and was repeated with the fractions. Results of quantitative phytochemical analysis showed the alkaloids to be the most abundant in the crude extract (34.30±0.14 mg/100g) followed by phenols (23.19±0.12 mg/100g). Cardiac glycosides had the least amount (3.89±0.04 mg/100g). Others were saponins (18.10±0.05 mg/100g), steroids (13.74±0.19 mg/100g), flavonoids (16.49 ±1.08 mg/100g), terpenoids (7.45±0.10 mg/100g) and tannins (9.72±0.25 mg/100g). Acute toxicity (LD50) value for the extract was found to be >5000 mg/kg body weight. Antimicrobial effects of the crude extract and that of the most effective fraction (F3) were also evaluated. The activities of the crude extract and fractions at different doses up to 800mg/kg body weight were compared with that of the standard drug, loperamide (0.5 mg/kg). Data were analyzed by the software, statistical package for the Social Sciences (SPSS), Version 18.0. Results obtained showed that the crude extract had significant antimicrobial activity at 200%, eliciting zones of inhibition of 10.20±0.85, 12.00±3.30, 9.60±0.72, and 10.37±1.72 mm against the test isolates Klebsiella pneumonia, Shigella flexneri., Citrobacter feundii and Salmonella paratyphi respectively. In the in vivonti-diarrhoeal studies, the crude extract at all doses used showed significant (P<0.05) antidiarrhoeal activity evidenced by delay in the onset of diarrhea of up to 56.00±4.36 mins when compared with the control 58.60±3.65. The extract also significantly decreased the distance travelled by the charcoal meal in dose- dependent pattern with activities which compared favourably with that of loperamide (P>0.05). Reduction in the intraluminal fluid accumulation in the castor oil-induced diarrheal model was also observed in all animals treated with the extract. All fractions (F1-F6) at 800mg/kg bodyweight of the leaves of Gouania longipetala exhibited significant (P<0.05) antidairreal activity but fraction 3 (F3) had the highest activities in the castor oil induced diarrhea, enteropooling and charcoal meal transit time. GC-MS spectral analysis of the ethanol extract of Gouania longipetala revealed twenty compounds including spartein, kamferol, oleic acid, 2-tetradecanol, dodecanoic acid, 1-octadecene, dodecanoic acid, 5,6-dehydrolupanine, propanoic acid, 3-chloro methyl ester, luparine, sapogenin A, catechin, flavon-3-ol, anthocyanin, resveratrol, linoelaidic acid, anagyrine, methyl 9,12-heptadecadienoate, baptifoline and ethyl oleate. The biological activities of each compound were discussed in present attempt. The molecular docking assay revealed Sapogenin A to possess an analogous feature with loperamide, which suggests that the antidiarrheal activities of Gouania longipetala could be due to the presence of Sapogenin A. The results of this study showed that Gouania longipetala has a significant (P<0.05) antidiarrhoeal effect and could be seen as a potential source of a new anti-diarrhoeal agent.
TABLE
OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables xi
List of Figures xiii
List of Plates xiv
List of Abbreviations xv
Abstract xvi
CHAPTER ONE: INTRODUCTION 1
1.1 Background of the Study 1
1.2 Aim of the Study 3
1.3 Objectives of the Study 3
1.4 Statement of the Problem 4
1.5 Justification for the Study 4
CHAPTER
TWO: LITERATURE REVIEW 6
2.1 The Gastrointestinal Tract 6
2.1.1 Basic functions of the gastrointestinal tract 7
2.1.2 Gastrointestinal motility 7
2.1.2.1 Gastrointestinal secretion and absorption 7
2.1.2.2 Gastrointestinal as a barrier 8
2.1.3 Gastrointestinal pathophysiology 9
2.1.3.1 Gut immune system 10
2.1.4 Flavonoids and gastrointestinal tract 10
2.1.4.1 Gastrointestinal tract and flavonoid metabolism 11
2.1.4.2 Gastrointestinal tract and flavonoids in health
and disease 12
2.2 Diagnosis 17
2.2.1 Treatment for ibs 19
2.2.2 Inflammatory bowel diseases 20
2.2.2.1
Pathogenesis of Ulcerative colitis 21
2.2.2.2
Crohn’s disease 22
2.3 Diarrhea: A Disorder of the Gastrointestinal
Tract 25
2.3.1 Acute diarrhea 25
2.3.1.1 Epidemiology and etiology 26
2.3.1.2 Pathogenesis of acute diarrhea 26
2.3.1.3 Clinical assessment 27
2.3.1.4 Laboratory evaluation 27
2.4 Management Approaches for Diarrhea 28
2.4.1 Fluid therapy 28
2.4.2 Antidiarrheals 28
2.4.3 Antiemetics 30
2.4.4 Antimicrobials 30
2.4.5 Prevention 30
2.5 Electrolyte Transport Dysbiosis in
Diarrhoeal Disease 31
2.6 Mechanisms of Action of Anti-Diarrhoeals 36
2.6.1.1 Mechanism of action of probiotics 37
2.6.2.1 Mechanism of action of antimotility agents 41
2.7.0 The Plant Gouania longipetala 41
2.8 Loperamide
(immodium) 44
2.8.1 Mechanism
of action of loperamide 48
CHAPTER THREE:
MATERIALS AND METHODS 49
3.1 Materials 49
3.1.1 Instruments/equipments 49
3.1.2 Materials 49
3.1.3 Chemicals 50
3.2.1 Collection
of plant materials and authentication 51
3.2.2 Preparation
of Gouania longipetala leaf extract 51
3.3.1 Animals 52
3.3.2 Microorganisms 52
3.4.1 Qualitative phytochemical screening of the
extract 52
3.4.2 Quantitative phytochemical screening of the
extract 54
3.5 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of the Extract 58
3.6 Acute Toxicity (LD50)
Evaluation of Gouania longipetala crude
Extract 59
3.7 In vivo evaluation of the effect of gouania
longipetala extracton
charcoal meal transit
in rats 59
3.8 Effect
of Gouania longipetala extract on
castor oil-induced diarrhoea
in rats 60
3.9 Effect of crude extract of Gouania
longipetala on castor oil-induced
fluid accumulation and serum electrolyte concentrations
in rats 62
3.9.1 Estimation of serum electrolytes 63
3.10 Bioassay-guided Fractionation of the
Ethanol Extract of gouania longipetala
leaf exract 66
3.10.1 Preparation
of sample slurry 66
3.10.2 Packing of
the glass column 67
3.10.3 Gradient
elution solvents 68
3.10.4 Thin- layer chromatography (TLC) 70
3.10.4.1
Plate preparation for thin-layer
chromatography 69
3.10.4.2 TLC
solvent systems and detection 69
3.11 In
vitro evaluation of anti-microbial activity of gouania longipetala leaf
crude extract and
fractions 73
3.11.1 Preparation of stock solution of gouania
longipetala leaf crude extract
and most bioactive fraction 73
3.11.2 Reactivation
of stock cultures of test organisms 73
3.11.3 Inoculation
of test organisms 73
3.11.4 Test for anti-microbial
activity 73
3.11.5 Determination
of minimum inhibitory concentration 74
3.12 Procedure for
Fourier Transform Infrared (FTIR) 75
3.13 Docking Procedure
and Analysis 75
3.14 Statistical Analysis 76
CHAPTER FOUR: RESULTS AND DISCUSSION 77
4.1 Results 77
4.1.1 Qualitative
phytochemical analysis of gouania
longipetala 77
4.1.2 Quantitative phytochemical analysis of gouania longipetala
leaf extract 79
4.1.3 Acute toxicity evaluation of Gouania longipetala leaf extract 81
4.1.4 Antimicrobial
activities of Gouania longipetala 84
4.1.5 Castor-oil
induced diarrhea 87
4.1.6 Castor oil- induced enteropooling 91
4.1.7 Assay of fractions on castor oil- induced
diarrhea 93
4.1.8 Assay of fractions on castor oil- induced
enteropooling 97
4.1.9 Measurement of charcoal meal transit time 101
4.1.10 Evaluation of fractions of the extract on
charcoal meal transit time 104
4.1.11 Estimation
of serum electrolytes for castor oil-induced antidiarrhoeal
Model 107
4.1.12 Evaluation of effects of fractions and crude
extract on electrolyte levels 109
4.1.13 Result of GC-MS analysis of the crude extract 111
4.1.14 Result of molecular docking of Gouania longipetala leaf extract 116
4.2 Discussion 122
CHAPTER FIVE: SUMMARY
AND CONCLUSION 131
References 133
LIST OF TABLES
3.1: Solvent
proportion 69
3.2 Pooling of fractions 72
4.1: Qualitative
phytochemical composition of Gouania
longipetala leaf
extract 78
4.2: Quantitaive
phytochemical composition of G.
longipetala leaf extract 80
4.2 Result
of acute toxicity evaluation of Gouania
longipetala leaf extract 80
4.3: Phase 1
result of acute toxicity evaluation of Gouania
longipetala leaf
extract 82
4.4: Phase 2 result of acute toxicity
evaluation of Gouania longipetala
leaf extract 83
4.5: Diameter
of zones of inhibition of the ethanol extract of
gouania longipetala
on the test bacteria isolates (mm) 86
4.6: Effect of the ethanol extract of gouania longipetala on castor
oil-induced diarrhea 89
4.5 Castor
oil- induced enteropooling 90
4.6 Assay of fractions on castor oil- induced
diarrhea 92
4.7: Effect of ethanol extract of leaves of Gouania longipetala on castor
oil-induced enteropooling in experimental albino rats 92
4.8: Effect of six different fractions and the
crude extract of
Gouania longipetala leaves on castor oil- induced diarrhea in
experimental rats 94
4.9: Effects of six different fractions and
crude extract of Gouania longipetala
on castor oil induced
enteropooling in experimental albino rats 99
4.10: Effect
of the ethanol extract of the leaves of Gouania
longipetala on
charcoal
meal test intestinal transit time 103
4.11: Effect of six different fractions and crude
extract of leaves of
Gouania longipetala
on charcoal meal intestinal transit time 106
4.12: Effects of the ethanol extract of the leaves
of Gouania longipetala
on concentration of electrolytes in the intestinal
contents 108
4.13: Effect of six different fractions and crude
extract of the leaves of
Gouania
longipetala on
concentration of electrolytes in the
intestinal contents 110
4.14: GC-MS spectral analysis of ethanolic extract
of leaves of
Gouania longipetala 112
4.15: Binding
affinities of the best ligand poses with specific macromolecules 118
LIST
OF FIGURES
4.1: Inhibition of stooling frequency in rats
following treatment with
graded
doses of ethanol extract of Gouania
longipetala and also
the
standard drug (Loperamide). 90
4.2: Percentage
inhibition in stooling frequency following treatment with
the the crude
extract and fractions of Gouania
longipetala leaves
extract and also
the standard drug (Loperamide). 96
4.3: Inhibition
of enteropooling 100
4.4: 2D molecular interactions of best docking
models of ligands vs macromolecule(6PT3). 119
LIST OF
PLATES
2.1: Gouania longipetala plant photographed
at a bush in Nsukka,
Enugu State, Nigeria 43
2.2: Gouania longipetala plant photographed
from a bush in Nsukka,
Enugu State, Nigeria 43
LIST OF ABBREVIATIONS
AAP - American
Academy of Pediatrics
AMPs – Antimicrobial
proteins
CD – Celiac
disease
CNS – Central
nervous system
COX – Cyclooxygenase
CRC - Colorectal
cancer
DPP-IV – Dipeptidylpeptidase
DRA - Down
Regulated in Adenoma
EC- Epithelial
cells
EEC – Enteroendocrine
cells
ETEC – Enterotoxigenic
E. coli
EIEC - Enteroinvasive
E. coli
FTIR – Fourier
transform infrared spectroscopy
GALT - gut-associated
lymphoid tissue
GCMS – Gas
chromatography and mass spectroscopy
GERD - gastroesophageal
reflux disease
GLP 1 and 2 – Glucagon-like-peptide 2
GIP - G-insulinotropic
polypeptide
HPA - hypothalamic
pituitary axis
HIC - High-income
countries
HIV – Human
immunodeficiency virus
IBD – Inflammatory
bowel disease
IBS – Inflammatory
bowel syndrome
IECs – Intestinal
epithelial cells
IL- Interleukin
JAMs - Junctional
adhesion molecules
JAK - Janus
kinase
LABs – Lactic
acid bacteria
LMIC - low-
and middle-income countries.
LPS – Lipopolysaccharides
mRNA – Messenger-RIBONUCLEIC
ACID
TGFβ1 – Transforming
growth factor beta1
ORT – Oral
rehydration therapy
PCR - Polymerase
chain reaction
PAT - Putative
Anion Transporters
TJ – Tight
junctions
T2D – Type
2 Diabetes
TLR4 – Toll-like
receptor 4
TNFα – Tumour
necrotic factor alpha
UC – Ulcerative
colitis
WHO – World
Health Organization
CHAPTER
ONE
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Diarrhoea is a major cause of
mortality and morbidity among children worldwide (Mokomane et al., 2018). It is the passage of three or
more loose, liquid or unformed stools per day, or more frequently than is
normal in an unchecked or uncontrollable frequency for an affected animal or
individual. It is predominantly a symptom of gastrointestinal infection- a
clinical condition referred to as gastroenteritis- which can be elicited by a
number of bacterial, viral and parasitic organisms. Infection is commonly due
to poor hygiene and is widely spread through contaminated food or drinking
water, or from person to person (WHO, 2013). Besides the infection-associated
causes of diarrhoea, a number of non-infectious causes include lactose
intolerance, irritable bowel syndrome, non-celiac gluten sensitivity (Sapone et
al., 2012), celiac disease,
inflammatory bowel disease (e.g., ulcerative colitis), hyperthyroidism, bile
acid diarrhoea (Slattery et al., 2015)
and some number of medications such as purgatives (Dekel
et al., 2017).
Severe diarrhoea leads to dehydration, and may pose a life-threatening
situation, especially in young children and the malnourished or immune-compromised
persons. According to WHO report (2017), diarrhoeal disease is the second
leading cause of death in children under five years old, and has on yearly
basis accounts for the deaths of approximately 525, 000 children. Diarrhoea can
persist for several days, and rid the affected individual’s body of fluid
(water and salts) that are necessary for survival. As such, these fluids and
electrolytes (sodium, chloride, potassium and bicarbonate) when lost through
liquid stools, vomits, urine and sweat, need to be urgently replaced.
Dehydration being one of the most severe threats implicated in diarrhoeal
disease occurs when these losses are not immediately replenished. For most
people, serious dehydration and fluid loss were the main causes of diarrhoea-
related deaths. Now, other factors such as septic bacterial infections are
steadily becoming more prevalent, accounting for an increasing proportion of
all diarrhea-associated deaths. Children who are malnourished or have impaired
immunity as well as individuals with HIV infection are most at risk of
life-threatening diarrhoea.
Diarrhoeal disease can be categorized
into three:
(a)
Short- term watery diarrhoea,
which involves only watery discharge, lasts only a few hours and sometimes goes
into days. Cholera typifies this type of diarrhoea especially in developing
countries and rarely in developed climes.
(b)
Short- term bloody diarrhoea,
which is also temporal, except that blood accompanies fluid loss. When blood is
present, it is also called dysentery.
(c)
Persistent diarrhoea – lasts 14 days or longer, and can
either be watery or bloody.
Most of these deaths occur in low- and middle-income
countries (LMIC). In high-income countries (HIC), the disease is rarely fatal,
but it is a leading cause of visits to the emergency department and
hospitalizations. While the major prevalence of severe diarrhoea, particularly
mortality, is in developing countries the impact of this disease is not limited
to developing countries (Liu
et
al., 2013). The
scourge of diarrhoeal disease owing to its repeated episodes is also a common
cause of malnutrition especially in those younger than five years of age. The
scourge of diarrhoea disease does not only pose short- term debilitating problems
but also other long- term problems which include stunted growth and poor
intellectual development (DuPont, 2014).
Most
pediatric diarrhea occur in the hinterlands, where the management and treatment
depend on the use of herbal medicines. For a couple of decades now, the
application of herbal medicines for treating ailments is rapidly taking the
centre stage, because they have little or no contraindications, are easy to obtain, and the cost implication
is negligible in contrast to modern
medicine. This work was proposed having in mind the invaluable importance of
medicinal plants to global healthcare.
1.2 AIM OF THE STUDY
The aim of this study is to evaluate
the antidiarrhoeal potential of ethanol extract and fractions of Gouania longipetala leaves.
1.3 OBJECTIVES OF THE STUDY
The
objectives of the study were to:
1.
Determine the phytochemical
composition of ethanol ectract of Gouania
longipetala leaves.
2.
Determine the LD50
of ethanol extract of G. longipetala leaves.
3.
Evaluate by GC-MS,
molecular docking and FTIR, the bioactive components in Gouania longipetala crude extract and the fraction with the highest
anti-diarrhoeal action.
4.
Evaluate in vivo antidiarrhoeal activities of Gouania longipetala crude ethanol
extract and fractions via charcoal meal transit studies in rats.
5.
Evaluate the inhibitory
effects of Gouania longipetala crude
ethanol extract and fractions in different models of castor oil- induced diarrhoea
in rats.
6.
Evaluate the effect of Gouania longipetala crude extract and
fractions on serum electrolyte contents in castor oil- induced diarrhoea in
rats.
7.
Evaluate the antimicrobial
effects of Gouania longipetala crude
ethanol extract and fractions against some diarrhoea causing microganisms.
8.
Establish by molecular
docking, the basis for the antidiarrhoeal effects of Gouania longipetala crude ethanol extract and fractions and hence
propose the mechanism(s) involved.
1.4 STATEMENT OF THE PROBLEM
Although synthetic chemicals as
anti-diarrhoeal agents are currently in use, they have such pitfalls as constipation,
abdominal discomfort, headache, dry mouth, nausea, and vomiting (Komal-Kumar and Rana, 2013). The use of traditional approaches remains the solution to the
undesirable contraindications posed by the conventional anti-diarrhoeal
medications. Alternative and traditional treatment approaches to diarrhoea include
the use of herbs in the forms of infusions, decoctions, and enema (Afolanya and
Wintola, 2014).
Other
alternative and complementary approaches include massage, acupressure, and
acupuncture. Some advantages derived from the application of traditional
medicine in the treatment of diarrhoeal include tolerability, high safety
profile, affordability, acceptability, accessibility, and wide spread
availability. Although a number of alternative medicines posed limitations such
as lack of dosage instruction, toxicity associated with some wild herbs, risk
of interaction with other conventional drugs, and lack of regulation, the
search for natural alternative anti-diarrhoeal compounds still remains the current
scientific goal. The objectives of discovering these alternative potential
anti-diarrheal agents can be achieved through laid down experimental procedures
(Buzescu, 2011). There is shortage of
detailed and updated techniques in the screening of promising agents as
alternative medicines in the treatment and management of diarrhea.
1.5 JUSTIFICATION FOR THE STUDY
According to the World Health
Organisation (WHO), the application of complementary medicine or alternative
medicine to treat infectious and non infectious diseases and disorders would
provide best sources from which new varieties of medications could be
developed. The traditional application of this specific plant Gouania longipetala for the treatment
and management of a plethora of health-related dysfunctions including, but not
limited to diarrhoea, underpins this study, which is designed to evaluate the
antidiarrhoeal potential of ethanol extract and fractions of Gouania longipetala leaves.
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