ABSTRACT
The objective of the present study was to evaluate the antidiarrheal activity of the methanol
stem bark extract of Hymenocardia acida in some laboratory animals. They methanol
stem bark extract of Hymenocardia acida (MEHA) was prepared from the freshly collected
stem bark of the plant. The stem bark was collected from Galadimawa village, Giwa local
government area of Kaduna State. The plant material was identified and authenticated by a
botanist in the herbarium section of the Department of Botany Faculty of Life Sciences
Ahmadu Bello University Zaria Nigeria and a voucher specimen (1275) was deposited for
future reference. The plant material was washed, shade dried, pulverized and sieved to
obtained fine powder. The powdered plant material was extracted with 70% v/v methanol
by cold maceration for three days with occasional shaking and agitation. The mixture was
then filtered using whattman No 1 filter paper and the filtrate was later concentrated over a
water bath at temperature of 40 to 450C to obtained dried extract. The dried extract was
kept in a bottle until needed for used. Preliminary phytochemical screening was carried
out using thin layer chromatography analysis (TLC). Acute toxicity study was carried out
using the method described in Organization for Economic Co-operation and Development
(OECD) guidelines 423. Invivo antidiarrheal screening of the extract at the doses of 600,
300 and 150 mg/kg body weight was conducted using castor oil induced diarrhea model,
castor oil induced enteropooling and gastric transit in mice model. In vitro study was
carried out on isolated rabbit jejunum and guinea pig ileum using ugo basile
microdynamometer, at extract concentration of (8 x 10-2 to 640 x 10-2 mg/ml) and
thereafter interacted with spasmogens; acetylcholine and histamine at concentration of (2 x
10-5 to 16 x 10-5). Alkaloids, glycoside, saponin, tanins, triterpenes, flavonoids, and
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steroids were detected from the extract. The result of the acute toxicity study revealed the
LD50 value to be in excess of 2000 mg/kg. The methanol stem bark extract of
Hymenocardia acida at all doses investigated significantly (p≤ 0.05) and dose dependently
delayed the onset of diarrhea in all the investigated groups in castor oil induced diarrhea
model compared to the control group. The extract at the doses of 300 and 600 mg/kg also
reduced the frequency of diarrheal feces compared to the negative control group.
Significant reduction (p≤0.05) in the propulsive movement and transit of charcoal meal
was observed at 600 mg/kg dose of the extract in gastric transit time in mice model. All
extract treated groups produced significant reduction (p≤0.05) in the volume of fluid
accumulation compared to the deionized water treated group in fluid accumulation test.
The methanol stem bark extract (8 x 10-2 to 640 x 10-2 mg/ml) produced significant
reduction in the tone and rate of spontaneous contraction of rabbit jejunum and relaxes
guinea pig ileum. The extract upon interaction with the spasmogens; acetylcholine and
histamine (2 x 10-5 to 16 x 10-5) inhibited contraction induced by both Acetylcholine and
histamine in a concentration dependent manner. The result of this study suggests that the
methanol stem bark extract of Hymenocardia acidapossess antidiarrheal activity that
justified its ethnomedical use in the treatment of diarrhea by herbal practitioners.
TABLE OF CONTENTS
Title Page ………………………………………………………………………………………………………………. ii
Declaration …………………………………………………………………………………………………………… iii
Certification ………………………………………………………………………………………………………….. iv
Acknowledgment ……………………………………………………………………………………………………. v
Abstract ……………………………………………………………………………………………………………….. vi
Table of Contents ………………………………………………………………………………………………… viii
List of Figures ……………………………………………………………………………………………………… xii
List of Tables ………………………………………………………………………………………………………. xiii
List of Appendices ………………………………………………………………………………………………. xiv
List of Abbreviations, Glossary and Symbols ……………………………………………………………. xv
CHAPTER ONE ……………………………………………………………………………………………………. 1
1.0: INTRODUCTION …………………………………………………………………………………………… 1
1.2 Justification of study ………………………………………………………………………………………… 3
1.3 Aim and Objectives ………………………………………………………………………………………….. 4
1.3.2 Specific objectives …………………………………………………………………………………………… 4
The specific objectives of the present work are to: ………………………………………………………. 4
1.4 Hypothesis ……………………………………………………………………………………………………….. 5
CHAPTER TWO …………………………………………………………………………………………………… 6
2.0 LITERATURE REVIEW …………………………………………………………………………………. 6
2.1 Diarrhea ……………………………………………………………………………………………………………. 6
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2.1.1 Definition ………………………………………………………………………………………………………. 6
2.1.2 Aetiology ……………………………………………………………………………………………………….. 6
2.1.3 Epidemiology …………………………………………………………………………………………………. 8
2.1.5 Risk Factors for Diarrhea ……………………………………………………………………………….. 10
2.1.6 Types of Diarrhea ………………………………………………………………………………………….. 10
2.1.7 Complication of diarrhea ………………………………………………………………………………… 13
2.1.8 Prevention of diarrhea ……………………………………………………………………………………. 14
2.1.9 Management of diarrhea …………………………………………………………………………………. 15
2.1.10 Antidiarrheal drugs ………………………………………………………………………………………. 17
2.2 The Plant Hymenocadia Acida …………………………………………………………………………… 20
2.2.1 Botanic description ………………………………………………………………………………………. 20
2.2.2 Biology ………………………………………………………………………………………………………. 21
2.2.3 Taxonomy …………………………………………………………………………………………………… 21
2.2.4 Synonyms ………………………………………………………………………………………………….. 22
2.2.5 Ethno-Medicinal Uses of Hymenocardia acida …………………………………………………. 23
CHAPTER THREE …………………………………………………………………………………………….. 24
MATERIALS AND METHODS …………………………………………………………………………… 24
3.1 Materials ……………………………………………………………………………………………………….. 24
3.1.1 Plant material ………………………………………………………………………………………………… 24
3.1.2 Experimental animals …………………………………………………………………………………….. 24
3.1.3 Equipment ……………………………………………………………………………………………………. 24
3.1.4 Chemicals and drugs ……………………………………………………………………………………… 25
3.2 Methods ………………………………………………………………………………………………………….. 25
3.2.1 Preparation of plant material …………………………………………………………………………… 25
3.2.2 Thin layer chromatography (TLC) analysis ………………………………………………………. 25
3.2.3 Acute toxicity studies. ……………………………………………………………………………………. 26
3.2.5 In vivo antidiarrheal studies ……………………………………………………………………………. 27
3.2.4 In vitro studies ………………………………………………………………………………………………. 29
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3.2.6 Data analysis ………………………………………………………………………………………………… 30
CHAPTER FOUR ……………………………………………………………………………………………….. 31
4.0 RESULT ………………………………………………………………………………………………………… 31
4.1 Percentage Yield of Methanol Stem Bark Extract of Hymenocardia acida ……………… 31
4.2 Phytochemical Constituents of Methanol Stem Bark Extract of Hymenocardia acida . 31
4.3 Toxicity Profile (LD50) of Methanol Stem Bark Extract of Hymenocardia acida ……… 33
4.4 Castor Oil Induced Diarrhea in Mice ………………………………………………………………….. 35
4.5: Effect of Methanol Stem Bark Extract of Hymenocardia acida on Gastric Transit
Time in Mice ………………………………………………………………………………………………………… 37
4.6: Effect of Methanol Stem Bark Extract of Hymenocardia acida on Fluid
AccumulationTest (Enteropooling) ……………………………………………………………………. 39
4.7: Invitro Studies of Methanol Stem Bark Extract of Hymenocardia acida on Isolated .. 41
Tissues …………………………………………………………………………………………………………………. 41
4.7.1: Effect of Methanol Stem Bark Extract of Hymenocardia acida on Isolated …………. 41
Rabbit Jejunum ……………………………………………………………………………………………………… 41
4.7.2: Effect of Methanol Stem Bark Extract of Hymenocardia acida on Isolated Guinea
Pig Ileum ……………………………………………………………………………………………………………… 45
CHAPTER FIVE …………………………………………………………………………………………………. 49
5.0 DISCUSSION ………………………………………………………………………………………………… 49
6.0 SUMMARY, CONCLUSION AND RECOMMENDATION ……………………………. 57
6.1 Summary ……………………………………………………………………………………………………….. 57
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CHAPTER ONE
1.0: INTRODUCTION
In spite of the advent of various modern drug discovery and screening techniques,
traditional knowledge systems have given clues to the discovery of valuable drugs
(Damodar et al., 2011).
Diarrhea is the passage of three or more loose or liquid stools per day (or more frequent
passage than is normal for the individuals). It is an alteration in normal bowel movement
characterized by an increase in the water content/volume, frequency of stools and/or
abdominal pain. It involves both an increase in motility along with increased secretions
and a decrease in absorption (absorptive capacity of the intestine exceeded) of fluid and
thus loss of electrolytes particularly, sodium and water (WHO, 2013).
Diarrheal diseases are the second leading cause of death in children under five years old,
and is responsible for killing around 760 000 children every year (Ahmed et al., 2014).
Diarrhea can last several days, and can leave the body without the water and salts that are
necessary for survival. Children who are malnourished or have impaired immunity as well
as people living with HIV are most at risk of life-threatening diarrhea (Ahmed et al., 2014)
About 1.7 – 5 billion cases of diarrhea occur yearly with majority of the cases in
developing countries (WHO, 2013). In 2012, it is the second most common cause of death
in children younger than fives (WHO, 2013). In 2015 it accounted for 9 percent of all death
among children under five years old, this translated to over 1400 young children dying
each day or about 526,000 a year (UNICEF, 2016).
2
In many rural communities in the developing world, herbal medicines are almost always
the only readily accessible and affordable therapies for the control of many diseases
including diarrhea (Njume et al., 2009; Green et al., 2010). Up to 80% of developing
world rely on medicinal plantsfor primary health care needs (Payyappallimana, 2010).
1.1 Statement of Research Problem
Diarrhoea has long been recognized as one of the important health problems all over the
world, especially in developing countries (Rajamanickam et al., 2010). It is one of the
most common causes of morbidity and mortality, affecting mainly infants and children
(WHO 2010; Nicholaset al., 2016).
Although diarrheal disease is preventable and treatable it remains the second cause of
death in children under age of five. Globally, there are nearly 1.7 billion cases of diarrheal
disease every year (Ahmed et al, 2014). In 2012 it is the number one cause of death in
children under five accounting for the death of about 2195 everyday, more than AIDs,
malaria and measles combined (UNICEF, 2012). The disease is responsible for over a
quarter of death of children in the world in 2012 (Yilgwan and Okolo 2012). Most of these
deaths occur in developing countries where an estimated 25% of under-fives mortality is
directly attributed to diarrhea disease (Yilgwan and Okolo, 2012). In Nigeria over two
hundred thousand children died from pneumonia and diarrhea in 2015 (Emeka, 2015)
The drugs used for treatments of diarrhea are not completely free from side effects ranging
from mild to severe including, dryness of the mouth, drowsiness, dizziness, severe pain in
stomach, abdominal bloating and vomiting, constipation, anxiety, confusion, depression,
severe headache and muscle spasm (Brunner, 2010).
3
Plant extract can be important source of natural drugs for treatment of diarrhea. Medicinal
plants have been used in treatment of diarrhea and have demonstrated conventional
properties that need to be further investigated (Sarin et al., 2013).
Hymenocardia acida stem bark is widely used in traditional medicine for treatment of
diarrhea without any scientific validation.
1.2 Justification of study
Despite advancement in modern medicine and the various drugs available for treatment of
diarrhea including loperamide, bismuth subsalicylate and oral rehydration salt (ORS),
diarrheal diseases remains a major cause of mortality especially in developing countries
(WHO, 2013).
The use of herbal drugs in treatment of diarrhea is a common practice in many African
countries; in fact more than 80% of people in rural African communities still rely on
indigenous medicine as a primary source of health care (Agunu et al., 2005). This is partly
due to the high cost, difficult accessibility associated with modern health care system, and
sometimes conservative’s attachment to culture and tradition (Maroyi, 2011).
WHO encourages inclusion of herbal medicines of proven safety and efficacy in the
healthcare programmed of developing countries (WHO, 2002), in addition WHO also
encourages studies for the treatment and prevention of diarrheal disease depending on
traditional medical practice (Atta and Mouneir, 2004).
4
Generally, it is known that anti-diarrhea plant extracts have antispasmodic properties,
delay gastrointestinal transit, suppress gut motility, stimulate water absorption and/or
reduce electrolyte secretion (de Wet et al., 2010).
Scientific research is therefore needed to provide evidence of the safety and efficacy of
beneficial medicinal plants. The plant Hymenocardia acida stem bark is often used in
Nigeria for treatment of diarrhea (Amom et al., 2013). However, scientific basis for it use
has not been proven. Thus the present study was prompted to evaluate the anti-diarrhea
effect of the plant.
1.3 Aim and Objectives
1.3.1 Aim
The aim of the study was to evaluate the antidiarrheal activity of the methanol stem bark
extract of Hymenocardia acida in laboratory animals.
1.3.2 Specific objectives
The specific objectives of the present work are to:
i. Determine the acute toxicity profile of the methanol stem bark extract of
Hymenocardia acida
ii. Evaluate the effects of the extract on castor oil induced diarrhea
iii. Evaluate effects of extract on castor oil induced enteropooling in mice.
iv. Evaluate the effects of the extract on gastric transit time in mice.
v. Evaluate the effect of the extract on perfused isolated rabbit jejunum and guinea pig
ileum.
5
1.4 Hypothesis
The methanol stem bark extract of Hymenocardia acida possesses antidiarrhoeal activity
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