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ABSTRACT

The herbalists in several African Countries have used Rothmannia longiflora decoctions as
febrifugal and analgesic agent. The present study examined the analgesic, anti-inflammatory
and toxicological profile of the methanolic extract of the plant in experimental animals.
The preliminary phytochemical screening of the methanolic leaf extract of R.longiflora was
carried out and revealed the presence of cardiac glycoside, steroids/terpenoids, tannins,
saponins, flavonoids and carbohydrates.
The acute toxic effect (median lethal dose values (LD50) of methanolic leaf extract of
Rothmannia longiflora was carried out using Lork’s method in intraperitoneal and oral routes
in mice and rats and were found to be above 5000mg/kg body weight.
The anti-nociceptive effects of the extract was studied using acetic acid induced writhing test
in mice, hot plate test in mice and formalin induced pain in rats. The anti-inflammatory effect
was studied using Carrageenan induced paw oedema in rats.
The extract at doses of 50-1000mg/kg significantly (P<0.05) inhibited the number of acetic
acid-induced abdominal writhes in mice dose dependently. The highest inhibition of
abdominal constriction (64.9%) observed at 1000mg/kg was greater than that of piroxicam
(61.5%), the standard non-steroidal analgesic and anti-inflammatory drug used in the study.
The methanolic leaf extract at doses of 250-1000mg/kg R. longiflora significantly (P<0.05)
and dose-dependently protected the mice against thermally induced pain stimulus in mice but
there was no significant thermal protection with the dose of 50mg/kg.
The 500 and 1000mg/kg doses of R.longiflora extract offered more than 100 and 200%
respectively while the standard drug (Morphine sulphate) offered more than 300% protection
against thermally induced pain stimulus in mice.
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There was no significant inhibition of both the neurogenic pain (early phase) and
inflammatory pain (late phase) at 50mg/kg and 250mg/kg doses of the extract.
However, there was significant (P<0.05) inhibition of both the neurogenic (early phase) and
inflammatory (late phase) pain dose dependently at 500mg/kg and 1000mg/kg with highest
inhibition at 1000mg/kg (53.33%). The significant (P<0.05) inhibition of the standard drug
(Morphine sulphate) at the late phase was 46.67%; this doubled that of the early phase which
was 20%.
The methanolic leaf extract of R.longiflora significantly (P<0.05) inhibited Carrageenan
induced paw oedema at doses of 250mg/kg; 500mg/kg and 1000mg/kg dose-dependently.
There was no significant inhibition at dose of 50mg/kg.
The percentage anti-inflammatory effects at the peak of the Carrageenan-induced oedema at
the third hour were; 39.39%, 51.52%, 63.63% at respective doses of 250mg/kg, 500mg/kg
and 1000mg/kg compared to 69.70% for Piroxicam 20mg/kg (the standard anti-inflammatory
drug)
Renal function test showed no statistically significant difference (P>0.05) of the levels of
Sodium (Na+), Potassium (K+), Chloride (Cl-) ions, Urea and Creatinine compared to that of
the control at the end of 30 days (sub-chronic) and 90 days (chronic) studies.
In liver function test, there was no statistically significant difference (P>0.05) in the levels of
AST, ALT, and ALP enzymes in all the treated groups compared to the control group after
the 30 days studies. However, there was statistically significant difference with 500mg/kg
and 1000mg/kg after the 90 days treatment.
The Histopathological study showed no observable gross lesions during the periods of subchronic
and chronic studies.
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These findings suggest that methanolic leaf extract of R.longiflora possesses analgesic and
anti-inflammatory potentials/effects that justify the ethnomedical use of the plant in the
treatment of painful and inflammatory conditions by the herbal practitioners. In addition, the
results of toxicity studies showed that the extract is non-toxic to the kidneys and the heart
during the sub-chronic and chronic toxicity studies however; mild fatty change was observed
with 500mg/kg in the liver after the chronic studies.

 

 

TABLE OF CONTENTS

LE PAGE
Title page————————————————————————————— i
Declaration————————————————————————————- i i
Certification ———————————————————————————— i i i
Dedication————————————————————————————— i v
Abstract—————————————————————————————— v
Acknowledgement—————————————————————————– v i i i
Table of contents—————————————————————————— i x
List of tables ———————————————————————————– x i i
List of plates ——————————————————————————— x i i i
List of appendices —————————————————————————- x v i i
Abbreviations ——————————————————————————– xviii
CHAPTER ONE
1.1 Background information ————————————————————— 1
1.2 Justification —————————————————————————— 2
1.3 Statement of problem——————————————————————– 3
1.4 Model for the studies——————————————————————– 4
1.5 Aims and Objectives of the Study ————————————————— 7
1.6 Research Hypothesis —————————————————————— 8
2.0 CHAPTER TWO
2.0 Literature review———————————————————————— 9
2.1 Analgesics——————————————————————————– 9
x
2.2 Prostaglandin———————————————————————- 1 2
2.3 Pain———————————————————————————– 1 3
2.4 Inflammation———————————————————————— 2 4
2.5 Kidney——————————————————————————- 2 6
2.6 Liver——————————————————————————— 2 6
2.7 The plant, R.longiflora———————————————————— 2 9
3.0 CHAPTER THREE
3.0 Materials and Method———————————————————- 3 2
3.1 Plant Material——————————————————————– 3 2
3.2 Animals————————————————————————– 3 2
3.3 Drugs and Chemicals———————————————————– 33
3.4 Equipment and other materials———————————————– 3 4
3.5 Preliminary phytochemical screening—————————————- 3 4
3.6 Lethal dose (LD50) determination——————————————– 3 9
3.7 Analgesic Studies—————————————————————— 3 9
3.7.1 Acetic acid induced writhing test in mice———————————— 3 9
3.7.2 Hot plate test method in mice————————————————– 4 0
3.7.3 Formalin test in rats————————————————————– 4 1
3.8 Anti-inflammatory studies——————————————————— 4 2
3.8.1 Carrageenan induced paw oedema ——————————————– 4 2
3.9 Toxicological studies————————————————————— 4 3
3.9.1 Sub-chronic toxicity studies—————————————————- 4 3
3.9.2 Chronic toxicity studies——————————————————— 4 3
3.9.3 Kidney function test————————————————————- 4 4
3.9.4 Liver function test————————————————————— 4 6
xi
3.9.5 Histopathological studies—————————————————- 4 7
3.10 Analysis of Data—————————————————————- 4 8
4.0 CHAPTER FOUR
4.0 Results—————————————————————————— 4 9
4.1 Preliminary phytochemical screening—————————————— 49
4.2 Results of lethal dose (LD50) determination———————————- 50
4.3 Analgesic studies—————————————————————– 51
4.4 Anti-inflammatory studies —————————————————– 5 5
4.5 Kidney function test————————————————————- 5 6
4.6 Liver function test————————————————————— 5 8
4.7 Histopathological studies——————————————————– 60
5.0 CHAPTER FIVE
5.0 Discussion ————————————————————————- 9 2
6.0 CHAPTER SIX
6.0 Conclusion and Recommendation——————————————– 99
REFERENCES———————————————————————– 1 0 1
APPENDICES———————————————————————— 110

 

 

CHAPTER ONE

INTRODUCTION
1.1 BACKGROUND INFORMATION
Traditional medicine is defined as “therapeutic practices that have been in existence, for hundreds of
years before the development and spread of modern sciences and are still in use (WHO, 2002).
Many people in the developing countries rely on traditional healing practices and medicinal plants
for their daily health care needs. This is due to the fact that traditional medicine is the most
affordable and accessible health care system available. Traditional medicine can therefore be
described as the total combination of knowledge and practices, whether explicable or not, used in
diagnosing, preventing or eliminating a physical, mental or social disease and which may rely
exclusively on past experiences and observations handed down from generations to generations,
verbally or written. The practice utilizes vegetables, animals and minerals, and other methods
(WHO, 1998). Results obtained from research into substances used in herbal medicine practices led
to discovery of relevant plants with useful chemical constituents that can be used in treatment of
various diseases (Amos et al,. 2001). A medicinal plant as defined by World Health Organization is
“any plant in which one or more of its parts contains substances that can be used for therapeutic
purposes or which are precursors for the synthesis of useful drugs” (WHO, 2002).
Quite a number of medicinal plants discovered by herbal practitioners have proved to be important
sources of potential therapeutic agents e.g. Quinine (antimalarial agent) from Cinchona bark,
digoxin and digitoxin (cardiac activity) from Digitalis purpurea, zingiberone (anti-helmintic) from
Zingiber officinalis, vincristine and vinblastine alkaloids (anti-leukaemic agents) from Catharantus
roseus, bourvadine from Bourvadin ternifolia, sinocaculine from Corculus tribolus, taxol from
Brevifolia used as anticancer agents etc. (Hardman, 1991)
1.2 JUSTIFICATION
xxi
Plants and plant products are extensively used in herbal medicine worldwide, as they are easily
available and cheap. Plants have served as valuable starting materials for drug development even in
advance countries of the world (Sofowora, A. 1993). Many people in the developing countries rely
on traditional healing practices and medicinal plants for their daily health care needs (WHO, 1998).
This is due to the fact that traditional medicine is the most affordable and accessible health care
system available.
Non-Opioid analgesics (NSAIDS) (examples naproxen, aspirin, ibuprofen) are particularly useful
for the treatment of low to moderate pains of muscular skeletal conditions but they exert serious side
effects on: GIT (abdominal pain, nausea, gastric ulcers, anaemia, GI perforations and
haemorrhage.). Renal/Hepatic (Salt and water retention, oedema, worsening of renal function in
renal/cardiac and cirrhotic patients, decrease effectiveness of anti-hypertensives and diuretics
medications), while prolonged use of paracetamol at high dose can cause liver damage (Goodman
and Gilman, 2006)
Opioid analgesics (examples, pethidine & morphine) are used usually to relieve moderate to severe
pain particularly of visceral origin. However, there are serious adverse effects such as: dependence,
tolerance, addiction, respiratory depression, hypotension, constipation, drowsiness, etc.
The adverse effects of both opioid and non-opioid analgesics couple with their high cost, limit their
uses by the patients in the management of pains hence the need to search for newer analgesics and
anti-inflammatory agents which are relatively safe, cheap, available, and potent.
This project therefore, seeks to investigate some of the pharmacological activities of methanolic
leaf extract of Rothmannia longiflora salisb plant to justify the traditional local uses particularly the
claim of febrifugal and analgesic properties, and to assess its toxic effects on the kidney and liver.
1.3 STATEMENT OF RESEARCH PROBLEM
Pain is the most common reason for physician’s consultation in various health care delivery systems
xxii
in Nigeria and in other countries of the world. Majority of clinical conditions associated with
humans are usually accompanied with pains, which can be mild, moderate or severe. Examples of
such disease conditions include; cancer, migraine, sickle cell anaemia, gouty arthritis, angina
pectoris. Others are; burns (severe or minor) and surgery (post-operative pain). Pain relievers
(analgesics) are often used as adjuncts in the therapeutic management of these conditions.
Uncontrolled abuse of analgesics and analgesic combinations may lead to renal damage severe
enough to cause end stage renal disease –ESRD- (Linton, 1980) or even the development of
urogenital cancer (Mohoney, et al, 1977). In United States, the cost of treating ESRD by either
dialysis or organ transplantation was $6.6 billion in 1991 (Inglehart, 1993). It has been estimated
that patients on therapy for ESRD due to analgesic abuse represent about 3% of cases in Queen land
Australia (Buckalow, 1986). In Nigeria such figures are scarcely available for the consumption of
the public and regulatory bodies.
Many plants have been identified for their analgesic and anti-inflammatory properties by various
researches on medicinal plants but most of these medicinal plants have not been subjected to studies
to ascertain their effects on these organs (stomach, kidney, Heart, and liver). Studies of the effects of
these plants on these organs will add more value to the efficiencies of these plants as analgesic and
anti-inflammatory agents.
1.4 THEORETICAL FRAMEWORK
In recent times, focus on plant research has increased all over the world, and more than 13,000
plants have been studied between 1996 and 2000 (Dahanukar et al., 2000).
1.5 MODEL FOR THE STUDIES
The models available for study of pain in experimental animals include:
Acetic acid induced writhing:
xxiii
This is a sensitive test for detecting substances with analgesic activity. In this test, pain is
induced by injection of irritants such as glacial acetic acid (0.6-0.7%) into the peritoneal cavity
of a mouse. The animal reacts by characteristic stretching behavour called writhing. Writhes can
be described as a wave of constriction and elongation passing caudally along the abdominal wall
with twisting of the trunk and extension of the hind limbs in mice. This is due to the nociceptive
property of acetic acid (Surender and Mafumdar, 1995). The writhing induced by chemical
substances is due to sensitization chemo-sensitive nociceptors by prostaglandins. The test is used
to access substances with analgesic properties acting through peripheral mechanism (Sutharson,
et al., 2007).
Hot Plate method
This method is described by (Lanhers et al (1992) and modified by (Mohamed and Ojewole (2004)
is employed. A 600ml test beaker is placed on a regulated thermostat hot plate at 550C±10C.
Each mouse is placed in the beaker (on the hot plate) and the response to electrical heat-induced
nociceptive pain stimulus is obtained. Licking of the paws or jumping out of the beaker is taken as
an indicator of the animals’ response to heat-induced nociceptive pain stimulus. The time taken for
each mouse to lick its paws or jump out of the beaker is noted (reaction time). Each mouse serves as
its own control. Before treatment, its reaction time is taken thrice at 1 hour interval and the mean
represents reaction time before treatment of the mouse. The mean reaction time of the groups is
pooled to obtain the final control mean reaction time (Tb). The test is used to access substances with
analgesic properties acting through central mechanism.
Formalin-induced pain:
This test is a chronic pain model which is sensitive to centrally acting analgesic agents
(Dubuisson and Dennis, 1977The test also differentiates between inflammatory and nonxxiv
inflammatory pains. Huskaar et al.,(1985) defines the distinct period of response to include the
early response (first phase) and the late response (the second phase). Drugs that act centrally e.g.
morphine inhibit both phases equally while peripherally acting drugs inhibit the late phase
(Shibata et al.,1989; Chan et al., 1995). The early phase is probably a result of stimulation of
nociceptors in the paw and reflects centrally mediated pain while the late phase is due to the
inflammation with release of serotonin, histamine, bradykinin and prostaglandins and at least to
some degree, the sensitization of central nociceptive neurons (Tjolsen et al., 1992).Activity in
this model suggest the activation of the opioids receptors (Gaertner et al., 1999).
Anti-inflammatory Activity Studies:
Carrageenan-induced paw oedema:
Carrageenan-induced hind paw oedema is the standard experimental model for acute
inflammation. Carrageenan is the phlogistic agent of choice for testing anti-inflammatory drugs
as it is not known to be antigenic and is devoid of apparent systemic effect (Chakraborty et al.,
2006). Moreover, the model exhibits a high degree of reproducibility (Winter et al., 1962). The
probable mechanism of action of Carrageenan-induced inflammation is bi-phasic, the first phase
is attributed to the release of prostaglandins and lysosome enzymes in 2 to 3 hours (Brooks and
Day, 1991).
Toxicology
Toxicology is concerned with the study of the harmful effects that chemical agents produce in living
organisms. Chemical substances may evoke one or both of two toxic effects. The first, an acute
effect, occurs shortly after contact with a single dose of poison. Chronic effect however occurs when
an organism is exposed to repeated small and non-lethal doses of a potentially harmful substance
(Hassel, 1982). The acute, sub-acute and chronic toxicity studies were carried out in this work.
xxv
1.6 AIM AND OBJECTIVES OF THE STUDY
1.6.1 Aim of the Study
The aim of this study is to scientifically investigate the analgesic and anti-inflammatory activities
and toxicity profile of the methanolic leaf extract of Rothmannia longiflora salisb plant in
experimental rats and mice.
1.6.2 Specific Objectives
(i) To collect and identify the plant Rothmannia longiflora salisb.
(ii) To extract the leaves of Rothmannia longiflora using pure methanol (100% v/v
methanol) as the solvent.
(iii) To conduct preliminary phytochemical screening on the methanolic leaf extracts of
Rothmannia longiflora salisb so as to identify the various chemical constituents
present.
(iv) To carry out Lethal Dose determination (LD50) on the methanolic leaf extract of
Rothmannia longiflora by Lorke’s method.
(v) To screen the leaf extract of R.longiflora for analgesic and anti-inflammatory
activities.
(vi) To investigate the toxic effects of the extract on the kidney, heart and liver.
1.7 RESEARCH HYPOTHESIS
Ho (Null hypothesis): The methanolic leaf extract of Rothmannia longiflora salisb contains active
constituents that are responsible for analgesic and anti-inflammatory activity which may cause renal,
cardiac and hepatic side effects.
Ha (Alternate hypothesis): The methanolic leaf extract of Rothmannia longiflora salisb does not
contain active constituents that are responsible for analgesic and anti-inflammatory activity which
may not cause renal, cardiac and hepatic side effects.

 

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