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

 

 

 

 

 

 

 

 

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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