Anti-Diarrhoeal Potential Of Ethanol Extract of Gouania longipetala Leaves
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 vivo anti-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 extract on
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
0.0
KENNETH, C (2023). Anti-Diarrhoeal Potential Of Ethanol Extract of Gouania longipetala Leaves. Mouau.afribary.org: Retrieved Sep 19, 2024, from https://repository.mouau.edu.ng/work/view/anti-diarrhoeal-potential-of-ethanol-extract-of-gouania-longipetala-leaves-7-2
CHIWUBA, KENNETH. "Anti-Diarrhoeal Potential Of Ethanol Extract of Gouania longipetala Leaves" Mouau.afribary.org. Mouau.afribary.org, 15 Aug. 2023, https://repository.mouau.edu.ng/work/view/anti-diarrhoeal-potential-of-ethanol-extract-of-gouania-longipetala-leaves-7-2. Accessed 19 Sep. 2024.
CHIWUBA, KENNETH. "Anti-Diarrhoeal Potential Of Ethanol Extract of Gouania longipetala Leaves". Mouau.afribary.org, Mouau.afribary.org, 15 Aug. 2023. Web. 19 Sep. 2024. < https://repository.mouau.edu.ng/work/view/anti-diarrhoeal-potential-of-ethanol-extract-of-gouania-longipetala-leaves-7-2 >.
CHIWUBA, KENNETH. "Anti-Diarrhoeal Potential Of Ethanol Extract of Gouania longipetala Leaves" Mouau.afribary.org (2023). Accessed 19 Sep. 2024. https://repository.mouau.edu.ng/work/view/anti-diarrhoeal-potential-of-ethanol-extract-of-gouania-longipetala-leaves-7-2