EFFECT OF MORINGA OLEIFERA LEAF EXTRACTS
ON THE ISOLATED RABBIT HEART AND UTERUS
BY
NTULUME R. DAVIS (BDS, MAK)
2007/HD11/9356U
A DISSERTATION SUBMITTED TO GRADUATE SCHOOL IN PARTIAL
FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF
MASTER OF SCIENCE IN PHYSIOLOGY OF MAKERERE UNIVERSITY
NOVEMBER, 2010
DECLARATION
I Ntulume R. Davis declare that this dissertation has never been submitted to any University for a
similar or any other academic award. All the information in this dissertation is based on my
observations.
SUPERVISORS:
1). Dr. Josephine Kasolo (MBChB, MSc (MAK)
Department of Physiology, Lecturer
Makerere University College of Health Sciences
Signature.............................................……...Date...........................................................................
2. Dr. Godfrey Bbosa (MSc, MAK)
Department of Pharmacology and Therapeutics, Lecturer
Makerere University College of Health Sciences
Signature....................……..........................Date...........................................................................
3. Dr. Isaac Okullo (BDS, ADHSM, MPH, PhD)
Department of Dentistry, Senior Lecturer
Makerere University College of Health Sciences
Signature....................……..........................Date............................................................................
ii
DEDICATION
I would like to dedicate this piece of work first to the Almighty God who has always seen me
through the difficult times of my education and indeed my whole life.
I also dedicate my work to my little girl Danny and my little boy Gabriel, I pray you two grow
up to be as bright as you so often seem to be.
iii
ACKNOWLEDGEMENT
I wish to express my sincere gratitude and acknowledgement to the following for all the
assistance they accorded me in my work;
First my family for praying for me, always encouraging me on and ever being so patient with
me. My colleagues at work who allowed me time off to work on my research and Mr. Aloysius
Lubega and Gordon Ewa in the Department of Pharmacology and Therapeutics, Ms. Teopista
Nayiga and Mr. Odoi Erisa in the Department of Physiology MUCHS, for all the assistance in
the experiments. I also acknowledge my supervisors for always being available to assist and
guide me in my research.
iv
TABLE OF CONTENTS
DECLARATION........................................................................................................................... ii
DEDICATION.............................................................................................................................. iii
ACKNOWLEDGEMENT ........................................................................................................... iv
TABLE OF CONTENTS ............................................................................................................. v
LIST OF FIGURES ...................................................................................................................... x
LIST OF TABLES ....................................................................................................................... xi
LIST OF ABBREVIATIONS .................................................................................................... xii
OPERATIONAL DEFINITIONS ............................................................................................ xiii
ABSTRACT ................................................................................................................................ xiv
CHAPTER ONE: INTRODUCTION ......................................................................................... 1
1.1 Background ........................................................................................................................... 1
1.2 Problem statement ................................................................................................................. 3
1.3 Research questions ................................................................................................................ 4
1.4 Objectives of the study .......................................................................................................... 4
1.4.1 General objective ....................................................................................................... 4
1.4.2 Specific objectives ..................................................................................................... 4
1.5 Justification of the study ....................................................................................................... 5
v
1.6 Significance of the study ....................................................................................................... 5
1.7 Conceptual frame work ......................................................................................................... 6
CHAPTER TWO: LITERATURE REVIEW ............................................................................ 7
2.1 Global use of traditional herbal medicine ............................................................................. 7
2.2 Burden of disease .................................................................................................................. 8
2.2.1 Burden of disease attributed to cardiac conditions .................................................... 8
2.2.2 Burden of disease attributed to obstetric conditions .................................................. 9
2.3 Physiology of uterine muscle contraction ........................................................................... 10
2.3.1 Plants with known effect on uterine function .......................................................... 12
2.4 Physiology of cardiac muscle contraction ........................................................................... 12
2.4.1 Plants with known effects on cardiac function ........................................................ 14
2.5 Moringa oleifera ................................................................................................................. 15
2.5.1 Uses of Moringa oleifera ......................................................................................... 15
2.5.2 The use of Moringa oleifera in obstetric care .......................................................... 17
2.5.3 The use of Moringa oleifera in management of hypertension ................................. 18
CHAPTER THREE: MATERIALS AND METHODS .......................................................... 19
3.1 Study design ........................................................................................................................ 19
3.2 Study setting ........................................................................................................................ 19
vi
3.3 Materials and equipment used ............................................................................................. 19
3.4 Preparation of experimental animals ................................................................................... 20
3.5 Selection criteria for the rabbits .......................................................................................... 20
3.5.1 Inclusion criteria ...................................................................................................... 20
3.5.2 Exclusion criteria ..................................................................................................... 20
3.6 Sampling procedure............................................................................................................. 20
3.7 Preparation of Moringa oleifera extracts ............................................................................ 21
3.7.1 Preparation of stock solutions .................................................................................. 22
3.7.2 Preparation of working dilutions of Moringa oleifera extracts and controls........... 22
3.8 Harvesting and preparation of the organs ........................................................................... 24
3.8.1 Preparation of the uterus .......................................................................................... 24
3.8.2 Preparation of the heart ............................................................................................ 25
3.9 Experiments on the organs .................................................................................................. 25
3.9.1 Experiments on the uterus ........................................................................................ 25
3.9.2 Experiments on the heart ......................................................................................... 26
3.10 Study variables .................................................................................................................. 26
3.10.1 Outcome variables ................................................................................................. 26
3.10.2 Independent variables ............................................................................................ 27
vii
3.11 Data collection and management ...................................................................................... 27
3.11.1 Kymograph recordings........................................................................................... 27
3.11.2 Data analysis and management .............................................................................. 27
3.11.3 Quality control ....................................................................................................... 27
3.11.4 Ethical considerations ............................................................................................ 28
CHAPTER FOUR: RESULTS .................................................................................................. 29
4.1 Effect of control solutions and leaf extracts of Moringa oleifera on the isolated rabbit
uterine muscle ........................................................................................................................... 29
4.2 Effect of control solutions and leaf extracts of Moringa oleifera on isolated rabbit cardiac
muscle........................................................................................................................................ 30
CHAPTER FIVE: DISCUSSION.............................................................................................. 33
5.1 Effect of Moringa oleifera leaf extracts on the rabbit uterine muscle ................................ 33
5.2 Effect of Moringa oleifera leaf extracts on the rabbit heart muscle ................................... 35
CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS ........................................ 37
6.1 Conclusion........................................................................................................................... 37
6.2 Recommendations ............................................................................................................... 37
6.3 Limitations of the study....................................................................................................... 37
REFERENCES ............................................................................................................................ 38
APPENDICES ............................................................................................................................. 44
viii
Appendix I: Composition of Locke’s physiological solution ................................................... 44
Appendix II: Checklist for uses of Moringa oleifera ................................................................ 45
Appendix III: Traces of effect of Moringa oleifera leaf extracts on the uterine muscle. ......... 46
ix
LIST OF FIGURES
Figure 1: Effect of varying concentrations of Moringa oleifera leaf extracts and control
solutions on the isolated rabbit uterine muscle ............................................................ 29
Figure 2: Effect of varying concentrations of Moringa oleifera leaf extracts and control
solutions on the contraction of isolated rabbit cardiac muscle ..................................... 30
Figure 3: Effect of varying concentrations of Moringa oleifera leaf extracts and control
solutions on the heart rate of isolated rabbit cardiac muscle ........................................ 31
Figure 5: Tracing of effect of Moringa oleifera leaf ethanol extract on the uterus ...................... 46
Figure 6: Tracing of effect of Moringa oleifera leaf aqueous extract on the uterus ..................... 46
Figure 7: Tracing of effect of Moringa oleifera leaf ether extract on the uterus .......................... 46
x
LIST OF TABLES
Table 1: Working solutions for Moringa oleifera leaf extracts and controls ............................... 23
Table 2: Comparison of the different Moringa oleifera leaf extracts and their effects on the heart
and uterus ....................................................................................................................... 32
xi
LIST OF ABBREVIATIONS
MAK
:
Makerere University, Kampala
MUCHS
:
Makerere University College of Health Sciences
M.O
:
Moringa oleifera
BP
:
Blood pressure
bpm
:
beats per minute
Hg
:
Mercury
doz
:
dozen
pcs
:
pieces
SA node
:
Sinoatrial node
PG
:
Prostaglandin
ATP
:
Adenosine triphosphate
cAMP
:
cyclic Adenosine monophosphate
FREC
:
Faculty Research and Ethics committee
CVD
:
Cardiovascular disease
DMSO
:
Dimethyl sulfoxide
ACH
:
Acetylcholine
TM
:
Traditional Medicine
xii
OPERATIONAL DEFINITIONS
Contraction of muscle
: The shortening or tensing of a part of a muscle or muscle fiber
Relaxation of muscle
: The act of lessening the tension in a muscle
Tension in a muscle
: The physical condition of being stretched or strained
Extracts
: Refers to Moringa oleifera leaf ether, ethanol and water extracts
Traditional medicine
: Diverse health practices, approaches, knowledge and beliefs
(WHO definition)
incorporating plant, animal, and/or mineral based medicines,
spiritual therapies, manual techniques and exercises applied
singularly or in combination to maintain well-being, as well as to
treat, diagnose or prevent illness.
xiii
ABSTRACT
Background: Local communities, world over prevent and treat indigenous diseases using local
herbal remedies. Moringa oleifera is one of the many plants used in herbal preparations in
Uganda for treating numerous human ailments such as malnutrition, cardiovascular diseases and
uterine conditions among others. Despite its wide use by communities around the world, there is
inadequate scientific information available on the actual pharmacological effects of Moringa
oleifera leaves on the heart and uterine muscles.
Study Objectives: The aim of this study was to determine the effect of ether, ethanol and
aqueous extracts of Moringa oleifera leaves on the isolated heart and uterine muscles.
Methodology: Air dried Moringa oleifera leaves were pounded and crude extracts were
prepared using ether, ethanol and distilled water as solvents. Stock solutions of the extracts were
prepared using distilled water. Dimethylsulfoxide was used to facilitate dissolution of the ether
extract. From the stock solutions, serial dilutions were prepared and applied to isolated rabbit
heart and uterine muscles and the effect on the heart rate and contraction of the muscles was
measured from kymograph recordings using a lagendorff’s perfusion apparatus. The effects of
the extracts were compared against standard solutions of acetylcholine, adrenaline, oxytocin and
salbutamol.
Results: All Moringa oleifera leaf extracts caused a reduction in the contractility of the heart
muscle with ether causing -40%, ethanol -28.3% and aqueous extract -46.2%. The negative
change observed in heart rate by ether was -5%, ethanol -23% and aqueous extract -9%. On the
contractility of the uterine muscle, the aqueous extract caused a +240% change while the ether
and ethanol extracts caused -100% and -36% change respectively.
Conclusion: The aqueous, ethanol and ether extracts of Moringa oleifera leaves contain
compounds that cause reduction in heart rate and relaxation of cardiac muscle and may have
xiv
medical use in management of cardiac conditions like hypertension. The herb may also be used
for obstetric conditions however pregnant women need to take the aqueous form of the herb
with caution as it may cause contractions leading to miscarriages and abortions.
xv
CHAPTER ONE: INTRODUCTION
1.1 Background
Herbal medicine is the oldest form of healthcare known to mankind and most cultures have long folk
medicine histories that include the use of plants. The WHO recognizes herbal medicines as a valuable
and readily available resource for Primary Health Care and it has endorsed their safe and effective use
(1). It however recommends that many herbal remedies still need to be studied scientifically while
recognizing the experience obtained from their prolonged safe use over the years in the treatment of
various conditions in both humans and animals, such as cardiovascular diseases (CVDs) and obstetric
conditions.
In recent decades, cardiovascular diseases have emerged as major causes of morbidity and mortality in
many countries with hypertension being the commonest (2). Hypertension is generally defined as an
abnormal persistently raised blood pressure greater than 140/90mmHg (3) and not due to physical
exercise. It is estimated that a sixth of the world population is afflicted by sustained high blood
pressure (4). Hypertension is a chronic disease and its management requires prolonged treatment
which is very expensive and often not accessible to poor individuals hence people resort to the use of
herbal medicines.
Obstetric conditions are also a common problem especially in developing countries and more so in the
rural areas. Obstetric conditions are those maternal conditions that are as a result of a mother being
pregnant or arise in the process of giving birth. These conditions can be direct or indirect (5). Direct
conditions occur before or within 6 weeks of delivery (such as haemorrhage, abortion, eclampsia and
sepsis) while indirect conditions include anaemia, malaria, hepatitis and cardiovascular disease. There
are also psychological stressors like puerperal psychoses, post-partum depression (baby blues), suicide,
and strong fear of pregnancy and childbirth resulting from obstetric complications, interventions or
1
cultural practices (6). The management of these conditions often involves the use of nonpharmaceutical approaches like exercise and nutrition but also to a large extent allopathic medicine.
Despite their proven efficacy, allopathic medicines are more expensive and are also presumed to be
associated with a lot of side effects and they are moreover not readily accessible to the majority of the
people who need them.
On the other hand, herbal remedies are seen as less expensive and less toxic (7). In Uganda, rural
people often have to travel long distances to reach the nearest health centre and here the
‘modern/western’ type of medicine is often out of stock or may be unaffordable. These hardships force
people to resort to their local remedies which are plentiful, available, and affordable and are already
known to be efficacious in managing certain illnesses. People are thus increasingly willing to manage
their medical needs by using complementary and alternative medicines like herbs (7). Among the
medicinal plants that are commonly used in management of various conditions is Moringa oleifera.
This herb has been reported in the management of cardiovascular disease such as hypertension and in
inducing abortion by women in India (8, 9).
Substances in Moringa oleifera which are similar to those derived from other herbs (for example
glycosides) have become a basis for some of the commercial medications used today for the treatment
of heart disease, high blood pressure, pain, obstetric and other medical problems such as diabetes,
cancer, and AIDS (10). Traditionally women use herbal preparations for the process of labour. Herbs
are also prescribed by traditional birth attendants and healers for various reasons including increasing
chances of having twins, correcting a mal-positioned fetus, anaemia in pregnancy among others (11).
In Uganda the practice of using herbs has been passed on for generations and it is used not only by
rural but also by many ‘modern’ women who continue using the herbal adjuncts in addition to their
regular antenatal and postnatal prescriptions. Moringa oleifera has been reported to be used by women
for contraception and as an abortifacient (8). Herbs such as Moringa oleifera are reportedly used for
2
treatment of painful uterus, inducing uterine contractions, management of retained placenta and post
partum bleeding among other conditions and have thus been found useful in obstetric care (12).
Despite their reported beneficial uses, herbs have also been reported to cause unexpected and
occasionally fatal outcomes. Moringa oleifera has been reported to cause abortion in rats (8). It has
also been reported that compounds similar to those present in Moringa oleifera (cardiac glycosides)
have caused toxicity and death (13). The efficacy of Moringa oleifera in management of hypertension
and obstetric care has not yet been fully scientifically researched despite the wide usage and this
underscores the importance of careful studies aimed at improving and regulating the use of herbs like
Moringa oleifera.
1.2 Problem statement
The professional medical management of hypertension and obstetric conditions is a costly undertaking
and often the low income rural communities are not able to afford the necessary prescription drugs.
These prohibitive costs of allopathic medicine when compared to the many nutritional and medicinal
attributes of Moringa oleifera have led to many locals resorting to the use of this herb in managing
common ailments like hypertension and to alleviate the pain of menstruation as well as in child birth.
However, while Moringa oleifera is reportedly used to alleviate menstrual pains, other reports
implicated the same herb in causing abortion in rats. This would appear paradoxical since the herb
would then appear to have both relaxation and contraction effects on the smooth muscle of the uterus.
The reports of toxicity and death linked to its effects on the heart clearly show how little is known
about the safety and efficacy of this herb. Even if it is believed to be useful in obstetric care and
management of hypertension, the actual effect of Moringa oleifera on the uterus and heart muscle has
not been fully investigated.
3
Presently there is insufficient scientific evidence available in literature to back up the use and efficacy
of this herb in the management of hypertension and alleviation of menstrual pains in individuals who
use it. Indeed there is minimal appreciation of the range of effects that Moringa oleifera may have on
the different body organs when consumed in its crude form, as mostly is the case. This has created a
problem of increasing the risks involved with un-intended actions of the herb when given to treat
particular ailments.
1.3 Research questions
(i) What is the effect of ether, ethanol and aqueous leaf extracts of Moringa oleifera on uterine
smooth muscle?
(ii) What is the effect of ether, ethanol and aqueous leaf extracts of Moringa oleifera on cardiac
muscle?
1.4 Objectives of the study
1.4.1 General objective
To establish the effect of leaf extracts of Moringa oleifera on the isolated heart and uterine smooth
muscle of the rabbit.
1.4.2 Specific objectives
1) To determine the effect of ether, ethanol and aqueous leaf extracts of Moringa oleifera on
the heart rate and contraction of isolated rabbit cardiac muscle.
2) To determine the effect of ether, ethanol and aqueous leaf extracts of Moringa oleifera on
the contraction of isolated rabbit uterine muscle.
4
1.5 Justification of the study
Moringa oleifera appears to have multi-system effects in the human body as shown by its numerous
reported uses. It has become a popular herb in the community even though there is insufficient
scientific evidence to explain and validate its apparent uses and efficacy. National surveillance
systems to monitor and evaluate its efficacy and adverse events are virtually non-existent in Uganda.
The unregulated or inappropriate use of traditional medicines and practices can have negative effects
upon the users and community and hence was important to carry out this study to establish the effects
of Moringa oleifera in the body particularly on the heart and uterus to guide the community that
consumes this herb and to promote its rational use as a medicament.
1.6 Significance of the study
The results from this study will add knowledge to existing literature about the pharmacologic effects
of Moringa oleifera on the heart and uterus. The data may be useful as a guide to those who utilise the
plant as food or medicine to now appreciate and be mindful of this herb’s other effects in the body
especially on the heart and uterus. The results will also be helpful to physicians who as they prescribe
their allopathic medications will appreciate better the effects of this herb on these organs and the
chances of drug interactions with the herb. The data may also be used to refine and improve on the
local and traditional use of Moringa oleifera for the management of cardiac and obstetric conditions.
5
1.7 Conceptual frame work
Toxins from
microbial
infections
Central and
Autonomic
Nervous Systems
Chemicals and
Drugs
Foods and medicinal
plants like Moringa
oleifera
Hormones and
endogenous compounds
like adrenaline
Hormones and
endogenous compounds
like Oxytocin
Functioning of the
Uterus
Contraction/ relaxation
of the Uterus e.g during
labour or menstruation
Normal Physiological
changes like activity
Heart
Contraction/
relaxation of the
Heart
This study however focused on the effect of Moringa oleifera extracts on the uterus and the heart
muscles.
6
CHAPTER TWO: LITERATURE REVIEW
2.1 Global use of traditional herbal medicine
Herbal remedies have played a fundamental role in the development of human medicines over the
centuries. Plant extracts have long been known to have effects on the normal functioning of the body
and in many cases they are used as herbs. The World Health Organization (WHO) estimates that in
Africa up to 80 percent of the population use traditional/herbal medicine (TM) for their Primary
Health Care (1). Indeed today, many patients especially those with chronic illnesses are turning to
herbs as adjuncts to their ‘modern’ treatments.
The world market for herbal medicines is steadily increasing each year and so is the expenditure on
this method of healthcare (14). In the USA, Australia and the United Kingdom by 1997 this
expenditure was well over US$ 2 billion each and the most popular herbal products were reported to
contain ginseng, garlic, Echinacea species. and St. John’s wort (14). TM is sometimes also the only
affordable source of health care especially for the world’s poorest patients. In many developing
countries research has shown that a course of antimalarial drugs can cost several dollars. In Uganda
this costs atleast $9 (nine United States dollars) (15) and yet per capita out-of-pocket health
expenditure amounts to less than US$ 6 per year (16). This forces patients either to abandon allopathic
medicines or purchase only part of a full dose and this eventually causes loss of efficacy for the drug
as well as drug resistance. On the other hand, herbal medicines for treating malaria are comparably
cheaper and may sometimes even be paid for in kind.
In developing countries, broad use of TM is often attributable to its accessibility and affordability (14).
In Uganda the ratio of TM practitioners to population is between 1:200 and 1:400 compared to a ratio
of 1:20,000 or worse for allopathic practitioners and the majority of these ‘western trained’ doctors
congregate in cities or other urban areas making it difficult for rural populations to access their often
7
more expensive services (14). This increased suffering has forced many patients to resort to their local
herbal remedies which are more accessible and at a much lower cost in terms of time and money spent
to receive them. Among these herbs is Moringa oleifera which like most other herbs has been used for
long and has been found non-lethal in usual dosages. It has been reported to cure many medical
conditions and is thus a popular herb not only in Uganda but in many other nations (17, 18). Because
of the increased usage of herbal medicines globally, the WHO instituted research guidelines for
evaluating the safety and efficacy of herbal/traditional medicines (TM) whose goal is to strengthen
research in the evaluation of the safety and efficacy of herbs, and to strengthen and promote the
rational use of herbal medicines (19).
2.2 Burden of disease
This is a mathematical measure of loss of healthy life-years due to disabling diseases in a country's
population. Disease causes ill-health and impacts on an individual’s well-being physically, socially,
emotionally, financially and occasionally may lead to death. The WHO periodically reports about the
global status of health and highlights the effect of different diseases on the general health of the world.
Almost in all parameters Africa and some Asian countries suffer more from both communicable and
non-communicable diseases compared to other continents (20). The non-communicable diseases like
cardiovascular diseases and obstetric conditions are gaining more recognition globally due to their
impact on good health and productivity of the nations (21). Cardiac conditions like hypertension and
maternal ill-health contribute a lot to the global burden of disease (20).
2.2.1 Burden of disease attributed to cardiac conditions
In Africa, with recent improvement in the control of malnutrition and infectious diseases,
cardiovascular diseases (CVDs) have emerged as major causes of morbidity and mortality in many
8
countries (22). Hypertension is by far the most common CVD followed by rheumatic heart disease and
cardiomyopathies (2).
Around the world, it is estimated that over 1billion people are affected by high blood pressure, a
burden that is rising even further (4). It was estimated that almost 25% of American adults have high
blood pressure (23) although newer data (2004) raised the figures even higher (24). In Africa, and in
Uganda particularly, a number of studies done have reported lower prevalence of hypertension than in
developed countries (25, 26). The causes of high blood pressure vary but according to National
Institutes of Health, the causes of high blood pressure may include narrowing of the arteries, an
increased volume of blood or a faster heart rate among others. High blood pressure might also be
caused by any medical problem however, for most of the time, the cause is not known (essential
hypertension) (3). It is estimated that in 2002 managing hypertension cost the United states
government US$ 47.2 billion (21) a figure which could cripple most developing economies like
Uganda. Therefore its management is very expensive to the ordinary poor people and as a result many
of the poor people use Moringa oleifera and other herbs to manage the CVD even when its efficacy in
not scientifically established.
2.2.2 Burden of disease attributed to obstetric conditions
The WHO developed estimates of maternal mortality and morbidity related to five direct obstetric
conditions: post-partum haemorrhage, puerperal sepsis, pre-eclampsia and eclampsia, obstructed
labour and abortion and the most common of these is the latter with an incidence of 14.8% globally
(6). The most common sequel of abortion was also found to be infertility.
Child bearing does not always end successfully and the loss of a fetus is a common reality in
pregnancy. In some cases this is a conscious and often illicit decision although it may also be
medically sanctioned. The causes of abortion are many and may be due to the uterine muscle per se,
9
hostile environment in the womb or factors related to the fetus which makes sustenance of life in-utero
impossible. It is estimated that about one in five pregnancies worldwide end in abortion and 29 in
every 1000 women of child bearing age (15-44 years) had experienced an abortion in 2003 (27).
However also, unsafe/illegal abortions happen more frequently in developing (Africa and Asia) as
compared to developed nations and in such cases abortions are procured either by using oral or
injectable medicines, trauma to the abdomen, introducing foreign bodies into the uterus or through
preparations inserted into the vagina (28). This may have teratogenic effects or cause uterine muscle
contractions that induce abortion. The effect of this is that over 5 million women worldwide are
admitted to hospital for abortion related reasons like sepsis, rupture of membranes with haemorrhage
and fatalities that account for 13% of maternal deaths annually (29). The many more women who
survive the experience of abortion often have to deal with related problems like infertility, chronic
morbidity and permanent physical and psychological impairment for much of the rest of their lives (6).
Regarding Moringa oleifera and its use for different ailments in various communities, the herb can
cause various effects on the body organs including the uterus. However, its effect on the uterus has not
been documented and scientifically evaluated.
2.3 Physiology of uterine muscle contraction
The contraction of the uterus is regulated by both the neural and the endocrine systems in the body.
The uterus is innervated by the autonomic nervous system through both sympathetic and
parasympathetic nerves (30). Sensory fibers from the body of the uterus pass via sympathetic nerves
(T10–l1) to cause contraction and from the cervix via parasympathetic nerves (S2–4) to cause relaxation.
The actual contraction of uterine smooth muscle also involves excitation-contraction coupling and
calcium ions however, smooth muscle has less well developed sarcoplasmic reticulum and hence
depends on the influx of calcium ions from the extracellular fluid (ECF) via voltage gated Ca2+
channels (31). The calcium ions bind to the protein calmodulin (32) (instead of Troponin) and the
10
complex activates calmodulin-dependent myosin-light-chain kinase which phosphorylates and
activates the enzyme myosin ATPase to hydrolyze ATP and provide energy to allow actin filaments to
slide over myosin and produce contraction.
Stretching of the uterus or irritation of its walls or the cervix is also known to initiate uterine
contractions (33). This is particularly important in a gravid uterus and is evident just before delivery
when the fetal movements become increased and are believed to contribute to initiation of parturition.
Uterine contractility can also be mediated by hormonal mechanisms. Estrogen and oxytocin produce
contraction and progesterone causes relaxation (34). The effects of these chemical substances are more
pronounced in the gravid uterus in which receptors for the substances have become up-regulated.
Estrogens increase uterine contractility partly because they increase the number of gap junctions
between adjacent uterine smooth muscle cells thus facilitating electrical conduction between cells (33).
Oxytocin, an endogenous hormone secreted by the neuro-hypophysis, exerts its contractile effects by
stimulating prostaglandin release but also oxytocin responsiveness is partially enhanced by
prostaglandin control of calcium mobilization (35). Prostaglandins and cortisol are also believed to
cause uterine contractions especially during parturition. The effects of these hormones are mediated
through chemical reactions in the body involving calcium and sodium ions especially, just like for
other muscle tissue.
Therapeutically oxytocin is used to induce labor and augment dysfunctional labor for conditions
requiring early vaginal delivery for example rhesus problems, maternal diabetes and preeclampsia
(36). It can also be used in management of incomplete abortion and in control of postpartum uterine
hemorrhage among other uses. Salbutamol and other β2 agonist drugs cause relaxation of smooth
muscle and are useful in the treatment of asthma while others are used as tocolytic agents in the
treatment of premature labor (36).
11
2.3.1 Plants with known effect on uterine function
There are a number of plants which are believed to affect uterine function and drugs have been
manufactured from these plants to modulate uterine function. The fungus Claviceps purpurea that
infects grains produces biologically active compounds called ergot alkaloids (37) which affect uterine
function. The synthetic preparations from this fungus include Lysergic acid diethylamide (LSD),
bromocriptine and ergotamine. For the uterus, ergot alkaloids in small doses induce rhythmic
contraction and relaxation of the uterus. At higher doses they induce prolonged and powerful
contractions which may lead to abortion.
The other group of agents is the eicosanoids which are mainly oxygenation products of polysaturated
long chain fatty acids. They are found in animals but also from plant sources (38). The main precursor
of these products is a 20-carbon fatty acid called arachidonic acid. Its products include prostaglandins,
thromboxanes and epoxides among others. Their uses include smooth muscle relaxation and treatment
of peptic ulcers (misoprostol derived from PGE1). In obstetrics, prostaglandins PGE2 and PGF2α cause
uterine contractions and may be used to initiate labour and also in first and second trimester abortions
(39). Among other medicinal plants reported to be used by traditional healers and local communities in
Uganda in pregnancy is Moringa oleifera and the herb is used to boost nutrition status and to manage
anemia (40).
2.4 Physiology of cardiac muscle contraction
Contraction of cardiac muscle is caused by electrical stimuli which are facilitated by chemical events
in the myocardial cells (41). The heart has an intrinsic ability to contract and the electrical signals
normally originate in centres called pacemakers of which the main one is the sino-atrial (SA) node
situated in the right atrium (42). The SA node generates an electrical impulse which travels through
the specialized conducting system of the heart thus stimulating first, the atria and then the ventricles to
12
contract. All these events are influenced by the parasympathetic and sympathetic nervous systems
which automatically regulate the heart rate and the velocity with which impulses are conducted
through the specialized conducting system of the heart (43). The sympathetic system releases
noradrenalin at corresponding nerve terminals and this increases the force of contraction and rate of
the heart beat while the parasympathetic system generally releases Acetylcholine at its nerve terminals
which generally slows down the heart. The two systems work together by physiological antagonism
and this way regulate the smooth normal functioning of the heart (41).
Noradrenalin and adrenaline are endogenous chemical substances that affect contraction of the heart
and it is from these that synthetic preparations are produced to mimic their effects in vivo.
Noradrenalin which is released from the adrenal medulla as a hormone but also from noradrenergic
neurons acts as a neurotransmitter in the central nervous system and sympathetic nervous system (44).
Methylation of the primary distal amine of noradrenalin by phenyl ethanolamine N-methyltransferase
(PNMT) in the cytosol of adrenergic neurons and cells of the adrenal medulla (so-called chromaffin
cells) leads to formation of adrenaline. Adrenaline is a non-selective agonist of all adrenergic receptors
in the body and increases the force of contraction (positively ionotropic) as well as the rate of
contraction of the heart (positively chronotropic) (44).
The neurotransmitter substance acetylcholine found in the parasympathetic system is on the other hand
both negatively ionotropic and chronotropic and causes relaxation of the heart (45). The contraction of
the heart allows it to pump blood into the vessels it supplies namely the aorta and pulmonary as well as
coronary vessels. The contraction of cardiac muscle is determined largely by the various ions found in
the cytoplasm of the myocardial cells. The main ions include sodium, calcium and potassium ions. The
sodium ions initiate the depolarization of the muscle cell membrane while the potassium ions are
mainly involved in repolarisation. Calcium ions maintain the plateau phase of the depolarization stage
but are also involved in the process of excitation-contraction-coupling by the interaction of calcium
13
ions from the sarcoplasmic reticulum of cardiac cells and actin-troponin-tropomyosin system in the
cardiac sarcomeres (46).
The coupling between excitation and contraction involves the transverse tubules system (T-tubules)
2+
which are associated with the sarcoplasmic reticulum and this contains a large store of Ca ions. When
a skeletal muscle fiber is excited, the depolarization of the sarcolemma is propagated into the T2+
tubules. Excitation of the T-tubules then leads to the release of Ca from the ends of the sarcoplasmic
2+
reticulum via so-called Ca
release channels ( ryanodine receptors) (46). The free calcium then binds
to Troponin-C (T-C) that is part of the regulatory complex attached to the myocardial filaments. When
calcium binds to the T-C, this induces a conformational change in the regulatory complex such that
Troponin-I exposes a site on the actin molecule that is able to bind to the myosin ATPase located on
the myosin head. This binding results in ATP hydrolysis that supplies energy for the movement
between the myosin heads and the actin causing them to slide past each other thereby shortening the
sarcomere length and hence contraction occurs (46).
2.4.1 Plants with known effects on cardiac function
There are a number of medicinal plants known to affect cardiac function and the most common in this
group are the cardiac glycosides mainly derived from the plant Digitalis purpurea (foxglove). Others
include the Apocynaceae family that includes Oleander and also Asclepias asperula which just like
Digitalis inhibit the Na+/ K+ ATPase pumps in heart muscle (47). The side effects of eating these
plants manifest as nausea, vomiting and cardiac arrhythmias. These plants have a positive inotropic
effect on heart function. They increase the intensity of the interaction of the actin and myosin
filaments of the cardiac sarcomeres thus causing contraction. They however have a low margin of
safety and are used cautiously in medical practice (48).
14
Other plants produce effects in the body that mimic the sympathetic system and are used for various
reasons as central nervous system (CNS) stimulants. They are also commonly abused and they include
Erythroxylum coca from which cocaine is manufactured and Cannabis sativa (Marijuana) which is
used therapeutically to provide analgesia and as an anti-emetic in cancer chemotherapy (49). In many
countries like Uganda the use of these drugs is outlawed. Stimulation of the CNS causes, among other
effects, a tachycardia and increased force of contraction of the heart. For Moringa oleifera, many
people in Uganda use this herb to manage CVD especially hypertension. However its effect on the
cardiac muscle is not known and documented despite its wide use.
2.5 Moringa oleifera
Commonly referred to as Moringa, this herb belongs to the family Moringaceae. It is a shrub or tree
that grows in a variety of forms depending on its geographical distribution. The genus Moringa
comprises over 15 species of plants of which Moringa oleifera has become the most widely cultivated
variety of the genus. The Moringa seeds originated from West Africa and in Uganda, the use of
Moringa oleifera became popular in the 1990’s (50). There are many popular beliefs and reported uses
among local communities world over about the uses of Moringa oleifera (18, 51-53). Almost every
part of the Moringa tree can be used as food, or has some other beneficial property. Among these
properties it has been reported that Moringa oleifera contains calcium, amino acids and other chemical
components that are known to affect muscle contraction (54-56). The different parts of the tree (leaves,
roots, bark, flowers, and seeds) are used in traditional medicine in the various countries where it is
found (17).
2.5.1 Uses of Moringa oleifera
Moringa oleifera is used as a micronutrient powder to aid in the treatment of many diseases (52). The
tree is a good source for calcium and phosphorus (54). In the tropics it is used as forage for livestock
15
(51, 57). Moringa oleifera leaves have been reported to contain more vitamin A than carrots, more
calcium than milk, more iron than spinach, more vitamin C than oranges, and more potassium than
bananas and the protein quality of Moringa oleifera leaves rivals that of milk and eggs (58, 59).
Lowell Fuglie in his work in Senegal and throughout West Africa reported that Moringa oleifera has
substantial nutritional benefit in many nutritional disorders (52). All essential amino acids were higher
than adequate concentration when compared with recommended amino acid pattern of the
FAO/WHO/UNO reference protein for a 2-5 year old child (60).
In a study on its anti-inflammatory activity, it was reported that Moringa oleifera has antiinflammatory activity (61) to the support of popular belief and traditional use of the herb in the
treatment for rheumatic and articular pain. This study reported that the aqueous extract of Moringa
oleifera roots indeed contained anti-inflammatory factors that could be useful in treatment of acute
inflammatory conditions (61). The hematological and hepato-renal functions of a methanolic Moringa
extract were also investigated in 1999 and it was reported that the crude extract decreased serum
aminotransferase and plasma cholesterol levels significantly (62). The total bilirubin, non-protein
nitrogen, blood urea and plasma protein were also altered. This is important because it is known that
cardiac function is closely related to renal function for example changes in the concentration of urea in
the blood have important effects on the concentration and reabsorption of the filtrate in the renal
tubules and eventually on the concentration and volume of the blood. A chronically elevated blood
volume leads to the process of volume-loading hypertension (63) which almost always causes injury in
the kidneys, producing many areas of renal destruction and, eventually, kidney failure, uremia and
death. This underscores the need for well controlled clinical studies which are still lacking and yet are
clearly of great value in guiding the consumption of this and other herbs.
Moringa oleifera leaf aqueous extract in low concentration is reportedly used for the regulation of
hyperthyroidism (64). In this study it was proposed that because of a decrease in active thyroid
16
hormone production when Moringa oleifera is used, the body is likely to have decreased general
metabolism. To postulate further, this means the tissues will have less need for nutrients found in the
blood and hence there would be decreased need for the heart to work hard in pumping blood to the
tissues. This would eventually lead to slowing down of the heart rate (bradycardia) and a decrease in
the strength of pumping.
Shukla et al. in 1988 also studied the effects of root aqueous extract of Moringa oleifera on the rat
uterus (65). They found that the extract caused failure of development of deciduoma and the
proliferation of vascular beds in the histo-architecture of the uterine lining which contributed to
alteration in the oestrus cycle leading to infertility. This is important because the plant despite being
useful in many ways may also be risky at certain doses and it underlines the importance of carrying
out research aimed at streamlining its utilization in humans and animals.
2.5.2 The use of Moringa oleifera in obstetric care
In developing countries like Uganda, especially in rural areas, many women do not deliver from
clinics and hospitals where there are skilled personnel and modern health services. In Uganda it is
estimated that only 42% of these women have skilled personnel attending to them at parturition (66).
The rest are mainly attended to by traditional birth attendants (TBAs) and relatives with rudimentary
knowledge of medicine. This also contributes to the high maternal death rate of 550 per 100,000 live
births in the country.
Due to the unavailability of allopathic medicines in such circumstances, herbal based remedies like
Moringa oleifera are used because they are readily available and cheaper (12). Moringa oleifera is
also believed to inhibit maintenance and growth of reproductive organs through hormonal action (67).
The herb is used by women in India as a contraceptive method and it has been shown to totally
inactivate or suppress the reproductive cycle (8).
17
2.5.3 The use of Moringa oleifera in management of hypertension
Hypertension is a chronic disease and its management is therefore very costly in terms of costs of
drugs and the distances and time patients have to travel especially in rural areas and this has made
people to resort to the use of herbal medicines. Moringa oleifera has been advocated as a remedy for
hypertension (18). It is argued that someone suffering from hypertension will benefit from Moringa
oleifera especially because of nutrients like calcium, magnesium, potassium, zinc and vitamins C and
E which the herb contains. Calcium is needed for muscle contractility and potassium for electrical
conductivity and ionic movements in cells. All these nutrients are found in high concentrations in
Moringa oleifera (56). It is known that increased level of sodium in the blood is a major contributing
factor to hypertension through increasing renal water retention and hence raising blood pressure (68).
The retention of sodium increases as the level of potassium in the blood decreases but Moringa
oleifera is a rich source of potassium and it is reported to contain 15 times more potassium content
compared to bananas (51). This high potassium content of Moringa oleifera may have effects (cause
lowering) on the membrane potentials of cells in the body and in the kidney may activate the Na +-K+
ATPase to prevent the excess reabsorption of sodium thereby keeping the blood pressure low.
Since systemic arterial pressure is directly proportional to cardiac output and total peripheral
resistance, it follows that hypertension may be caused by factors involving the vasculature, like
arthrosclerosis, but may also have origin from the heart like in cardiomyopathy and arrhythmias which
affect the cardiac output. Moringa oleifera as used for hypertension may therefore be having effects
directly on the cardiac muscle or the smooth muscle of the vasculature.
18
CHAPTER THREE: MATERIALS AND METHODS
3.1 Study design
This was an experimental study that involved testing of three separate Moringa oleifera leaf extracts
on the isolated heart and uterus of rabbits. The leaves of Moringa oleifera were used to make an ether,
an ethanol and a water extract to be used for the experiments.
3.2 Study setting
The study was conducted in both Makerere University College of Health Sciences Physiology and
Pharmacology and Therapeutics laboratories found in the Makerere University School of Biomedical
Sciences. The animals were kept in the Physiology laboratory while the experiments on the organs and
sample extraction were done in the Pharmacology and Therapeutics laboratory.
3.3 Materials and equipment used
-
Rabbits. (California breed)
-
Moringa oleifera dry leaves
-
Carbogen gas: made of 95% Oxygen and 5% carbon dioxide
-
Physiological solution: Locke’s solution.
-
Control reagents: Adrenaline, Acetylcholine, Salbutamol and Oxytocin
-
Kymograph: by C.F. Palmer (London) Ltd. Made in England
The contraction of muscles can be studied using a kymograph. This is a motor driven rotating drum
that can be operated at different speeds. The drum carries a calibrated graph paper that is written on by
a pen attached to various lever arms. These lever arms are connected using a thin light thread to the
heart muscle that is undergoing the experiment and when it twitches it moves the lever which then
leaves a mark on the paper.
19
3.4 Preparation of experimental animals
In this study, five male and five female California breed rabbits were purchased from the animal house
in department of Pharmacology of Makerere University College of Health Sciences. The sexes were
kept apart in rabbit pens for 2 weeks to acclimatize the animals to their environment. The animals
were fed on both fresh grassy and legume diet as well as rabbit pellet feed. At the start of the
experiments, the weight of the rabbits was determined using a standard weighing scale for rabbits. The
weights ranged from 1.6-2.4kg.
3.5 Selection criteria for the rabbits
3.5.1 Inclusion criteria
The study involved both male and female rabbits which were judged as healthy by having a record of
good appetite and clear solid stools. Their body temperature was measured using a body thermometer
placed in the rectum to ascertain that they were not febrile. It ranged from 37oC to 39oC. Three (3)
female rabbits along with three males were selected for the study. The male rabbits used were between
3-5months old and the female rabbits were at least 6months old with evidence from the breeder of
having had a litter previously.
3.5.2 Exclusion criteria
Any rabbit that displayed signs of ill-health like failure to feed, having diarrhea, a runny nose or being
febrile was eliminated from the study. Rabbits with external wounds and a rough coat depictive of
disease were also excluded from the study.
3.6 Sampling procedure
The desired outcome was to choose three hearts and three uteri for the study. To start sampling, the
five female rabbits were each tagged with a number written on a piece of paper, and with the help of
20
research assistants, paper lots were cast to select three from the group. All three female rabbits thus
selected were eligible for the study and were hence included. The same sampling procedure was done
to select 3 male rabbits from the group of 5. The ones not selected were returned to the pens. For
convenience, the uteri to be included in the study were chosen directly from the three selected female
rabbits (non-randomly) and the hearts were also selected non-randomly from the three selected male
rabbits.
3.7 Preparation of Moringa oleifera extracts
The plant samples were collected from the Makerere University Botanical Gardens. The plant samples
were correctly identified by a botanist and voucher specimen were also prepared and deposited at the
herbarium with reference number D001.
The identified Moringa oleifera samples were then air dried in a shade for 10 days till constant weight
was achieved. They were then pounded in a metallic mortar to fine powder for ease of extraction of
active compounds. The extraction procedures were carried out in the Department of Pharmacology and
Therapeutics laboratory of the College of Health Sciences, MAK. The process used followed the
already established serial extraction procedure of plant samples using ether followed by ethanol and
then distilled water as solvents (69).
Serial extractions were done using 185.6g of Moringa oleifera leaf powder got from the pounded plant
samples in an Erlenmeyer flask soaked in 400ml of ether for 3days with occasional shaking to
facilitate the extraction process. The mixture was then decanted and filtered using Whitman No.1
paper in a Buchner funnel. This process was repeated by soaking in ether for another 3days then
filtering again to increase on the yield. The residue was then air dried for 24hours in preparation for
ethanol (95% V/V) extraction following the same procedure in the same order as ether extraction.
Finally the residue was soaked overnight in 750mls of warm water for 10hours to prepare for aqueous
21
extraction. For water extracts, a freeze drier (Martin Christ 2- 1995 model) was used to evaporate the
water off while the ether and ethanol solvents were recovered using a rotary evaporator (Buchi
Rotavapor R-320).
3.7.1 Preparation of stock solutions
Different concentrations of the Moringa oleifera extracts were then prepared using the dry extracts
obtained from the residues. Concentrated stock solutions of 400mg/ml of the Moringa oleifera leaf
extracts were prepared using the dry extracts and distilled water as the solvent. This was done by
dissolving 2gm of each extract in 5ml of distilled water. The ether extract however required a few
drops of di-methyl sulfoxide (DMSO) to facilitate the dissolving process.
Stock solutions of the control solutions were also prepared. For salbutamol, 20mg of powder was
dissolved in 50ml of distilled water to form a stock of 0.400mg/ml. In the case of oxytocin, 21mg
(taken to be the average amount found in a vial of 10 international units) was dissolved in 50ml of
solution to make a stock of 0.420mg/ml, and 0.2mg of adrenaline was dissolved in 10ml of solution to
make 0.020mg/ml as a stock. Lastly the stock of Acetylcholine was made by dissolving 0.02mg in
10ml of distilled water to make 0.002mg/ml solution.
3.7.2 Preparation of working dilutions of Moringa oleifera extracts and controls
Using the stock solutions, serial dilutions were done to give a range of concentrations for use in the
experiments. Previously cleaned and dry test-tubes were set in a test tube rack and they were labeled
from the highest concentration obtained from the stock (400mg/ml in the case of the extracts) and then
each proceeding test tube had half the concentration value of the one before. 2mls of the stock solution
was drawn using a pippete and placed in the first test tube on the rack labelled 400mg/ml. 1ml of fresh
distilled water was similarly drawn using an unused pipette and placed in each of the proceeding
testtubes till the last one on the rack. 1ml of solution was then drawn from the first test tube with the
22
stock solution and this was placed in the second test tube to make a concentration that contained half
the original concentration in this test tube compared to the preceeding one. This sequence was
repeated for all following test tubes using 1ml from the preceeding test tube to be dissolved in the 1ml
of distilled water in the proceeding testtube to give a concentration that kept decreasing by half each
time. The concentrations used were thus as shown in the table below;
Table 1: Working solutions for Moringa oleifera leaf extracts and controls
Oxytocin
Salbutamol
Acetylcholine
Adrenaline
(mg/ml)
(mg/ml)
(mg/ml)
(mg/ml)
Moringa
oleifera extracts
(mg/ml)
0.42000
0.40000
0.00200
0.02000
400.00000
0.21000
0.20000
0.00100
0.01000
200.00000
0.10500
0.10000
0.00050
0.00500
100.00000
0.05250
0.05000
0.00025
0.00250
50.00000
0.02625
0.02500
0.00013
0.00125
25.00000
0.01313
0.01250
0.00006
0.00063
12.50000
0.00656
0.00625
0.00003
0.00031
6.25000
0.00328
0.00313
0.00002
0.00016
3.12500
0.00164
0.00156
0.00001
0.00008
1.56250
0.00004
0.78125
0.00002
0.39063
0.00001
0.19531
0.09766
0.04883
0.02441
0.01221
23
A similar procedure was also used for the control solutions with the first testtube having a
concentration equal to that of the stock solution it was drawn from and each preceding test tube having
half that amount as shown in the table above.
3.8 Harvesting and preparation of the organs
With the help of a skilled research assistant the animals were sacrificed one at a time by stunning with
a sharp blow to the back of the neck. The animals were then laid on their backs on a prepared
operating board. The underbelly was shaved and the skin carefully dissected to reveal the internal
organs. Periodic irrigation with freshly prepared Locke’s physiological solution was done to prevent
desiccation and death of the organs. The major vessels supplying the organs were ligated and then
severed to allow removal of the organs from the animals. This method was adopted from similar
methods from N.M Ghosh’s Fundamentals of Experimental Pharmacology (70).
3.8.1 Preparation of the uterus
About 3-5cm of the upper part of one horn of the uterus was resected from the main body of the uterus
and, attaching one end to the hook of a glass aerator tube and the other to the lever of the kymograph
using a thread sewn into the uterine wall, it was suspended in Locke’s solution at 38oC for about 10
minutes to allow it to relax first. Carbogen gas was continuously bubbled through the bath solution to
maintain good aeration and control pH of the solution. The kymograph cylinder speed was adjusted
and set to 24mm/min to start rotating and give a baseline tracing for 1-2 minutes before any agents
were added to the organ bath.
As a control, oxytocin first and then salbutamol which are known to cause contraction and relaxation
of these muscles respectively were added separately to the organ bath. The doses used for oxytocin
ranged from the smallest dose of 0.0016mg/ml to the largest dose 0.0525mg/ml where the scale was at
maximum while for salbutamol the doses ranged from the smallest 0.0016mg/ml till 0.400mg/ml. The
24
formula C1V1= C2V2 (where V1 was the known volume of the organ bath (20ml) and C1 the desired
concentration while C2 was the known concentration of the stock solution of the control) was used to
draw the volume V2 to balance the equation. The agents were promptly and thoroughly washed out
several times with warm physiological solution after each agent had been tested to re-establish the
baseline activity once a response had been recorded.
3.8.2 Preparation of the heart
The isolated heart was mounted onto a Lagendorff’s apparatus. The perfusion apparatus delivered
filtered Locke’s solution to a cannula inserted into the aorta of the rabbit heart so that the coronary
vessels were perfused at a pressure of about 40cm of water and at an average temperature of 38oC with
carbogen gas continuously bubbling through the reservoir. This kept the heart viable and beating.
A thread was attached using a small hook to the right ventricular wall of the heart and on the other end
to the lever of the kymograph whose pointer was adjusted to make a tracing on the kymograph paper.
Following the same process like for the uteri, adrenaline and acetylcholine in doses stated in the table
were injected via a side arm on the apparatus into the heart as controls. The continuous flow of
physiological solution promptly washed out the agents after each application and hence re-established
the baseline activity.
3.9 Experiments on the organs
3.9.1 Experiments on the uterus
Once the baseline activity had been re-established, different concentrations of the extracts were added
to the organ bath starting with the smallest dose. The observed effect (contraction/relaxation) was
recorded on the graph paper of the rotating drum. The extracts were tested following the order ethanol,
aqueous and lastly ether for the varying concentrations of the working solutions prepared. A fresh
uterus was used for each extract because one organ could not last the whole duration of the
25
experiment. The contact period of the extracts on the isolated uterus was timed to 3-5 minutes or until
a steady peak displacement was noted. The responses of the uterus obtained from the varying
concentrations of the extracts were noted and later the displacements were measured and recorded in a
log book and later on transferred to a computer for storage. The organ bath was emptied 2-3 times
thereafter and the liquid replaced with fresh warm physiological solution to wash out the extract
during the experimental period. At the time of washing out, the rotating drum was stopped so that any
movements happening in this period were excluded as being due to unintended contractions of the
organ muscle.
3.9.2 Experiments on the heart
Once the baseline activity had been re-established, the same procedure done for the uteri was followed
when testing for the heart responses. A fresh heart was again used for each extract and for the same
contact period. The effects that occurred were recorded on the kymogram tracing paper and afterwards
the displacements were measured with a ruler and again recorded in the log book and computer. The
extracts were washed out after a contact period of about 3-5 minutes and the liquid replaced with fresh
warm physiological solution. The heart muscle was also periodically irrigated on the external surface
to prevent drying out. This was done at regular intervals of about 2 minutes. The temperature of the
physiological solution was maintained at an average of 38°C.
3.10 Study variables
3.10.1 Outcome variables

Strength of contraction of the rabbit heart and uterus as recorded by the kymograph and
measured in millimeters of displacement and presented as a scatter graph of percentage change
in response against concentration applied.

Rate of contraction of the rabbit heart measured in seconds and presented as a scatter graph of
percentage change in response against concentration applied.
26

Description of the general trend of the response of the muscles to the applied extract.
3.10.2 Independent variables

Extracts of different concentrations
3.11 Data collection and management
3.11.1 Kymograph recordings
The displacement of the kymograph tracings away from the baseline was measured in millimeters for
various concentration strengths of the extracts and control solutions. This represented the strength of
contraction of the muscles which was either due to contraction or relaxation effect on the muscles. The
heart rate was recorded concurrently using a stop watch.
3.11.2 Data analysis and management
Data was entered into the computer using Microsoft Excel 2007 from where simple descriptive
analysis was done. The general effects of the extracts on the organs were described from the
observations. The relaxing and contracting effects of the Moringa oleifera leaf extracts were
calculated as a percentage of the induced tension which existed at the start of the experiment. The
results were presented in form of tables and scatter plots to show the trends and effects of the
concentration of extracts and controls applied to the organs.
3.11.3 Quality control
Quality control to increase accuracy of the data collected was done where an experienced research
assistant was involved in the preparation of the animals and calibration of the equipment for the
experiment. A pre-test experiment was done to sharpen the skills of the researcher and the assistant in
dissecting the rabbits and ensuring the organs stay viable during the course of the experiment. Sample
vouchers of Moringa oleifera were also prepared for purposes of verification and future identification.
All equipment used was cross-checked before use to check conformity with agreed standards like
27
calibration. The analytical grade materials used were purchased locally at the National Medical stores
and they were checked to ensure they were not expired. The temperature of the physiological solution
was kept at an average 38oC and the organs were tested with control reagent after each set of
experiments to see if the organs were still viable.
3.11.4 Ethical considerations
Permission to carry out the study was sought from the Department of Physiology and the Research and
Ethics Committee of the College of Health Sciences Makerere University, the School of Graduate
Studies of Makerere University and from the Uganda National Council for Science and Technology.
The ethical practices that govern laboratory animals like merciful killing and the right to food and
shelter were also adhered to.
28
CHAPTER FOUR: RESULTS
4.1 Effect of control solutions and leaf extracts of Moringa oleifera on the isolated rabbit uterine
muscle
Figure 1: Effect of varying concentrations of Moringa oleifera leaf extracts and control solutions
on the isolated rabbit uterine muscle
The graph shows that increasing the concentration of the aqueous extract led to an increase in the force
of contraction of the uterus. A similar effect was observed with oxytocin. The graph also shows that
increasing the dose of the ethanol and the ether extracts generally led to a decrease in the force of
contraction of the uterine muscle an effect similar to what was observed when salbutamol was used.
The graph shows that increasing the dose of the ether extract led to (100%) total cessation of
contraction at 0.390mg/ml of extract used. Effects of the crude extracts were also observed at higher
concentrations compared to those of the pure controls.
29
4.2 Effect of control solutions and leaf extracts of Moringa oleifera on isolated rabbit cardiac
muscle
Figure 2: Effect of varying concentrations of Moringa oleifera leaf extracts and control solutions
on the contraction of isolated rabbit cardiac muscle
The graph shows that increasing the concentration of all the extracts generally led to a decrease in the
strength of contraction of the cardiac muscle similar to the effect of acetylcholine. The decreasing
effect was more at high concentrations of the crude extracts as compared to the pure controls.
30
Figure 3: Effect of varying concentrations of Moringa oleifera leaf extracts and control solutions
on the heart rate of isolated rabbit cardiac muscle
The graph generally shows that increasing the concentration of the ethanol, the ether and the aqueous
extracts of Moringa oleifera leaves led to a decrease in the heart rate of the rabbit cardiac muscle. This
is similar to the effect of acetylcholine on the heart muscle.
31
Table 2: Comparison of the different Moringa oleifera leaf extracts and their effects on the heart
and uterus
Extract
Effect on heart (% change in
Effect on uterus (% change in
response) at 100mg/ml
response) at 0.391mg/ml
Heart Rate
Contraction
Contraction
Ether
-5
-40.0
-100
Ethanol
-23
-28.6
≈ - 36
Aqueous
- 9
-46.2
+240
The table shows that the ethanol extract has the greatest effect of reducing the heart rate while the
aqueous extract reduces the force of contraction more than the rest. On the uterus, the aqueous extract
causes increased force of contraction while the ether and ethanol extract decrease the force of
contraction of the uterine muscle.
32
CHAPTER FIVE: DISCUSSION
5.1 Effect of Moringa oleifera leaf extracts on the rabbit uterine muscle
The results show that increasing the concentration of the ethanol extract of Moringa oleifera leaves
caused a reduction in the force of contraction of the uterus as shown in figure 1. A similar trend was
observed when the same extract was tested on rat uteri (56). In comparison with the heart muscle in
figure 2 where 50% reduction occurred at 400mg/ml of the ethanol extract, in figure 1 the uterus was
50% relaxed at only 0.781mg/ml of the same extract. Since the same extract (and hence
phytochemicals) was applied on both organs, the results show that the uterus is much more sensitive to
the ethanolic extract than the heart muscle. This may be due to the fact that the two organs have
different receptors responsible for mediating the effects of the phytochemicals present in these extracts
or that particular phytochemicals require specific receptors for their action. The ethanol extract has
been shown to contain Glycosides, Terpenoids, Flavonoids, Saponins, Steroids and Tannins (71).
Flavonoids are known to be potent inhibitors of prostaglandin biosynthesis (72) and this could be the
cause of inhibition of the contractility of the uterus, however this requires further research to ascertain.
The aqueous extract caused a contraction of the uterine muscle with a big magnitude similar to that of
oxytocin as shown in figure 1. This effect could be attributed to calcium ions whose direct effect in
initiation of the contractile process cause effects opposite to those of potassium ions. However, the
same aqueous extract was also used for the heart and it caused relaxation therefore this raises
questions on the simple role played by potassium and calcium ions in the response of these muscles to
the extract. On the heart, the aqueous extract caused reduced force of contraction which was attributed
to more potassium ions and less calcium ions but on the uterus it was observed to cause increased
contraction. This means calcium ions are definitely in abundance as proposed in earlier research (54).
The result suggests that either the uterine receptors are very sensitive to the small quantity of calcium
33
ions in the extract or, there exists certain phyto-chemical substances which probably use multiple
mechanisms of action responsible for the contraction.
Furthermore, Moringa oleifera is consumed in the crude aqueous form by women in West Africa (73)
where it is reported to relieve the pain of menstruation; a process which would require relaxation of
the uterus and yet here the aqueous extract seemed to cause contraction. This paradox could be
because the concentration consumed by these women is seldom high enough to cause uterine
contractions or that the herb may possess both relaxing and contracting effects depending on the dose
consumed. It may also be due to different concentrations of the phytochemicals present in the extract
since it is known that the geographical location where the plant is grown affects the yield and
composition of plants (18). It is of particular interest to find that the aqueous extract caused 100%
increase in uterine contraction at only 0.024mg/ml. The result shows that this herb has notable effects
at small concentrations and stresses the need for caution in daily use. It may be appropriate to classify
Moringa oleifera as an abortifacient herb because in addition to causing physiological alterations of
the endometrium (74) which impair implantation of an embryo it has been shown here to cause
increased contraction of the uterine muscle as well and thus can induce abortion. However,
controversy still exists as to what exactly is responsible for the observed contraction and this requires
further research towards a more detailed analysis of this extract and its phytochemicals.
The ether extract caused a total cessation of even the intrinsic contraction of the uterine muscle as
shown in figure 1. There is a paucity of literature regarding the effect of ether extract of Moringa
oleifera leaves on muscle tissue. However, at the tissue level there must have been a total blockade of
the action potentials in the uterine muscle. This is probably through the effect of potassium ions
causing hyperpolarisation because subsequent addition of pure oxytocin re-established the contractility
and response of the muscle to even other lower concentrations of the extracts. The results show the
extract was not lethal to the tissue and probably only blocked ionic channels or depleted a neuro34
transmition agent however it leaves questions about what exactly happens to the cells themselves and
their organelles within. This requires further study to establish.
5.2 Effect of Moringa oleifera leaf extracts on the rabbit heart muscle
In figure 2 and 3, it shows that increasing the concentration of the ethanol extract of Moringa oleifera
leaves led to a decrease in the rate as well as the force of contraction of the heart muscle. Faizi et al
(1995) proposed that this effect is likely to be due to thiocarbamate glycosides found in the ethanolic
extract (9, 75). Thiocarbamate glycosides are thought to act by blocking calcium channels whose role
in muscle contraction is mentioned before and hence this will impair the contraction-excitation process
of the myocardium. The effects could also be due to potassium ions which have been shown to exist
abundantly in Moringa oleifera (51). Potassium ions in the extracellular fluid cause the heart muscle to
become flaccid which slows down the heart rate. This is partially because the potassium ions decrease
the resting membrane potential of the myocardial fibers thus progressively decreasing the intensity of
the action potentials in the muscle. Large quantities can even cause cardiac arrest by blocking
conduction of the impulse from the atria to the ventricles across the atrioventricular bundle in the
myocardium.
The aqueous extract of Moringa oleifera leaves also causes a reduction in the rate and contraction of
heart muscle as shown in figure 2 and 3. The reduction in the heart rate with the aqueous extract was
however smaller compared to the ethanolic extract. For the former, 100mg/ml caused a 9% reduction
compared to a 23% change for the latter. This shows that the ethanolic extract reduces the heart rate
more than the often consumed aqueous extract. Regarding contraction of the cardiac muscle, while
both extracts caused reduced contraction of the muscle, the magnitude of this effect (relaxation) was
found to be opposite to that for the heart rate. The aqueous extract at 100mg/ml caused a reduction of
46.2% compared to 28.6% of the ethanolic extract. It is not clear why these differences exist and this
will require further study however, this distinction is useful in cardiology since some diseases affect
35
the strength of the heart as a muscle pump (myocardial diseases) while others may interfere primarily
with the electrical sysytem of the heart rate to cause arrhythmias hence the need to develop drugs
specific for the particular problem.
In figure 2 and 3 the ether extract also revealed a similar trend as the ethanol and aqueous extracts and
increasing the concentration of the ether extract caused a reduction in the rate and force of contraction
of the heart muscle. It required 25mg/ml to cause 50% relaxation which was the smallest concentration
of the three extracts. It is possible that the ether extract contained more potassium ions compared to
the other extracts but it is also possible that the ether extract has the higher concentration of a
particular phyto-chemical substance/s responsible for relaxation of the heart muscle.
The apparent differences in response using similar concentrations of the extracts were also compared
in table 2. At 100mg/ml, the ethanol extract had the biggest effect in causing a reduction in the heart
rate while the aqueous extract reduced the contraction of the heart the most. At 0.391mg/ml, the
aqueous extract had the highest contractile effect on the uterus while the ether extract caused total
cessation of contraction at that concentration. Knowing that the different solvents used in extraction
have affinity for different chemicals, this result is likely to be due to dissimilar phyto-chemical
composition of the extracts as opposed to comparable potency.
36
CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS
6.1 Conclusion
The ether, ethanol and aqueous extracts of Moringa oleifera leaves contain compounds that reduce
heart rate and caused relaxation of cardiac muscle. These negative chronotropic and negative
ionotropic effects shown by the extracts on the heart are in support of popular use of this herb in
management of hypertension.
On the uterus the ether and ethanol extracts of Moringa oleifera leaves caused a reduction in the force
of contraction of the muscle. The aqueous extract however has compounds that caused uterine
contraction. These results show the potential of this herb for obstetric and cardiac applications.
6.2 Recommendations
1) The common prescription of Moringa oleifera for nutritional or other medical reasons should
be done cautiously taking into consideration the effects the plant has on the heart and the
uterus. Expectant mothers should be particularly careful as they consume the aqueous
preparation since it has been shown to cause uterine contraction which could lead to abortion.
2) The three solvents used in this research on Moringa oleifera leaves are known to extract
different phytochemicals from the plant. Further isolation and purification of the active
compounds should form the basis for further research to find out which of the phytochemicals
in the extract is responsible for the observed effects so as to open up possibilities for using this
herb in modern pharmaceutical practice.
6.3 Limitations of the study
Due to limitations in equipment it was not possible to do molecular studies to prove what exactly was
happening in the cells themselves and their surrounding environment which includes the ions in the
cytoplasm. This study could benefit from such further research.
37
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43
APPENDICES
Appendix I: Composition of Locke’s physiological solution
Component
Composition(g)
NaCl
0.900
KCl
0.042
CaCl2
0.024
NaHCO3
0.015
NaH2PO4
-
MgCl2
0.100
Dextrose
0.100
Distilled Water to
100ml
44
Appendix II: Checklist for uses of Moringa oleifera
Documented and reported use of Moringa oleifera leaf
Documented use of Moringa oleifera leaves
Use of Moringa oleifera leaves in Uganda
1. Dental caries
2. Urinary tract infection
3. Hesptein-Barr virus
4. HIV/AIDS ( Related symptoms)
5. Guinea-worm
6. Trypanosomiasis
7. Bronchiasis
8. External sores/ulcers
9. Fever
10. Hepatitis
11. Cancers prostate
12. Anti-hypertensive
13. Anti-anemic
14. Diabetes mellitus
15. Diuretic
16. anti- Thyroid
Worms
Skin disease
External sores/ulcers
Malaria
Anti-hypertensive
Diabetes mellitus
Colitis
Gastritis/ulcers
Anti-septic
Soap
Impotence
Syphilis
Flu
Asthma
Bone setting
Heart burn
18. Colitis
Energy
19. Diarrhea
20. Dysentery
21. Rheumatism
22. Gastritis/ulcers
23. Headache
24. Anti-septic
25. Catarrh
26. Tonic
27. Soap
35. Weight loss
36. Hypocholestrolemic
37. Antioxidants
38. Energy
39. Iron deficiency
40. Protein energy malnutrition
41. Vitamin/mineral deficiency
Protein energy malnutrition
Lactation enhancer
Worms in cattle
Stress
Source: Jedi, W. (2005) Moringa oleifera: A review of the medical evidence for its nutritional, therapeutic and
prophylactic properties. Trees of life Journal, (18).
45
Appendix III: Traces of effect of Moringa oleifera leaf extracts on the uterine muscle
Figure 4: Tracing of effect of Moringa oleifera leaf ethanol extract on the uterus
The tracing shows the reduction in contractility of the uterus as the concentration of Moringa oleifera
ethanol extract is increased from 12.2µg/ml to 781µg/ml.
Figure 5: Tracing of effect of Moringa oleifera leaf aqueous extract on the uterus
This tracing shows an increase in uterine contractility from 1.56mg/ml going higher to 12.5mg/ml and
the effect was similar with higher concentrations of the extract.
Figure 6: Tracing of effect of Moringa oleifera leaf ether extract on the uterus
The tracing shows a reduction in uterine contractility towards zero when the ether extract was used.
The tissue was challenged with oxytocin on the right side and it responded positively showing it was
still viable. Increasing the concentration of extract further led to total cessation of contraction.
46