ABSTRACT
Three Nigerian plants (Annona senegalensis, Azadirachta indica and Vernonia
amygdalina) were evaluated for antileishmanial effects in vitro and in vivo using the
Leishmania major parasites. It involved the preliminary testing of the hexane,
methanol and aqueous extracts of these plants on infected murine macrophages
and other cell lines. This was to determine the leishmanicidal activity and
cytotoxicity on normal cell lines (L929 fibroblasts and macrophages), cancer cell
lines (Jurkat and SH-5YSY) and Trypanosome brucei brucei. The leishmanicidal
activity was determined using the microscopic counting method while the
cytotoxicity involved the Alamar Blue colorimetric technique. Furthermore, doseresponse
sensitivity tests were carried out on bone marrow macrophages infected
with Leishmania major parasites and only extracts with positive indications from
the preliminary tests were used. Also, the efficacy of the extracts which gave
positive indications was tested in vivo in BALB/c mice treated orally for five days at
a dose of 50 mg/kg per day. The aqueous extract of Vernonia amygdalina
significantly (p<0.001) suppressed infection rate of intracellular amastigotes
(>60%) and significantly (p<0.001) reduced parasite levels to almost zero in the
infected macrophages. In the dose-response sensitivity test, the methanol extract
of Vernonia amygdalina suppressed infection rates (>50%) at a lower
concentration (50 g/ml) than that which caused toxicity to the macrophages. It
also significantly reduced the lesion sizes by about 50% as well as minimised
histopathological changes in the skin of infected mice. The hexane extract of
Annona senegalensis, showed good trypanocidal effect against Trypanosome
brucei brucei at a concentration of 3.13 g/ml. This was over 15 fold more potent
than the reference drug, cylemersan used at 50 g/ml.
viii
Also, the potentials of a DNA based vaccine and its recombinant protein were
evaluated as vaccine candidates. Leishmania donovani gene encoding gamma
glutamylcysteine synthetase and its soluble recombinant GCS antigen in the
presence of a non-ionic vesicular surfactant (NIVS) as an adjuvant were tested.
Susceptible BALB/c mice were immunised with the plasmid encoding the full
sequence for GCS (pVAXGCS), recombinant protein in presence of adjuvant
(rGCS-NIV) or plasmid alone (pVAX control) prior to challenge with a high dose of
Leishmania major promastigotes. Mice immunised with the recombinant protein
(rGCS-NIV), demonstrated an enhanced production of specific IgG1 and IgG2a
antibodies indicating that it was immunogenic. Also, it resulted in significantly lower
lesion sizes (p<0.05) as compared to the controls. This partial protection
corresponded to a significant increase (p<0.05) in gamma interferon production
suggesting an enhanced Th1 response. These results showed that the protein
vaccine from Leishmania donovani induced a strong immunoprotection against
cutaneous leishmaniasis, suggesting that it represents a very good candidate for
use as a vaccine against several leishmania species.
TABLE OF CONTENTS
Title page———————————————————————————————– i
Declaration———————————————————————————————ii
Certification——————————————————————————————–iii
Dedication———————————————————————————————iv
Acknowledgement———————————————————————————–v
Abstract————————————————————————————————vii
Table of contents————————————————————————————ix
List of tables—————————————————————————————–xv
List of figures—————————————————————————————-xvi
List of plates—————————————————————————————-xvii
Appendix——————————————————————————————–xviii
Abbreviations—————————————————————————————xix
CHAPTER ONE
1.0 Introduction———————————————————————————–1
CHAPTER TWO
2.0 Literature Review—————————————————————————6
2.1 Leishmaniasis——————————————————————————-6
2.1.1 Types of Leishmaniasis——————————————————————-7
2.1.2 Prevalence and distribution of Leishmaniasis—————————————8
2.1.3 Transmission and Life cycle of Leishmaniasis————————————–9
2.2 Cutaneous Leishmaniasis ————————————————————–12
2.3 Cutaneous Leishmaniasis in L. major infection————————————14
2.4 Immune responses to L. major infection——————————————–14
2.4.1 Role of T-cells—————————————————————————–14
2.4.2 Role of B-cells——————————————————————————18
x
2.5 Diagnosis of Cutaneous Leishmaniasis———————————————19
2.6 Histopathology of cutaneous Leishmaniasis—————————————20
Skin——————————————————————————————-20
Spleen—————————————————————————————-21
Lymph node———————————————————————————21
Liver——————————————————————————————-22
2.7 Treatment of Leishmaniasis————————————————————24
2.7.1 Chemotherapy—————————————————————————–24
2.7.2 Alternative Chemotherapy————————————————————–26
2.7.3 Immunotherapy—————————————————————————-27
Vaccines—————————————————————————27
Attenuated Vaccines———————————————————–28
Protein/Subunit Vaccines—————————————————–29
DNA vaccines——————————————————————–31
2.7.4 Phytotherapy——————————————————————————39
Plants used as antileishmanials————————————————-41
Quinones——————————————————————————41
Alkaloids——————————————————————————-42
Terpenes——————————————————————————-43
Phenolic Derivatives—————————————————————-44
Other Metabolites——————————————————————–45
2.7.5 Azadirachta indica————————————————————————48
2.7.6 Annona senegalensis——————————————————————–53
2.7.8 Vernonia amygdalina——————————————————————–56
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CHAPTER THREE
3.0 Materials and Method——————————————————————-60
PLANT EXPERIMENTS
3.1 Materials————————————————————————————-60
3.1.1 Animals and Parasites——————————————————————-60
3.1.2 Cell lines————————————————————————————-60
3.1.3 Chemicals, reagents and media——————————————————-60
3.1.4 Drugs—————————————————————————————–61
3.1.5 Equipment———————————————————————————–61
3.2 Methods————————————————————————————–62
3.2.1 Plant Collection—————————————————————————-62
3.2.2 Preparation of plant extracts———————————————————–64
3.2.2.1 Hexane and Methanol Extraction————————————————-64
3.2.2.2 Aqueous Extraction——————————————————————-64
3.2.2.3 Purification and Isolation of crude extract of V. amygdalina—————65
3.3 In Vitro Studies—————————————————————————–66
3.3.1 Macrophage Sensitivity Test ———————————————————-66
3.3.1.1 Isolation of Macrophages from bone marrow —————————-66
3.3.1.2 Infection of Macrophages——————————————————67
3.3.2 Assessment of drug toxicity————————————————————68
3.3.3 Antitrypanosomal Activity (Trypanosome brucei brucei)————————68
3.4 In Vivo Antileishmanial Test————————————————————69
3.4.1 Parasites and Infection——————————————————————69
3.4.2 Drug Treatment—————————————————————————-69
3.4.3 Tissue Processing————————————————————————70
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IMMUNOTHERAPY EXPERIMENT
3.5 Materials————————————————————————————-72
3.5.1 Animals and Parasites——————————————————————-72
3.5.2 Chemicals and Reagents—————————————————————72
3.5.3 Drugs—————————————————————————————–73
3.5.4 Bacteria and Protein———————————————————————-73
3.5.5 Equipment———————————————————————————–73
3.6 Methods————————————————————————————–74
3.6.1 Plasmid Vaccine Construction and Purification————————————74
3.6.2 Cloning of GCS for Protein Expression———————————————75
3.6.3 Expression and Purification of Recombinant Protein—————————-76
3.6.4 Analysis of Protein————————————————————————77
3.6.4.1 Gel Electrophoresis (SDS-PAGE)————————————————77
3.6.4.2 Western Blot Analysis————————————————————–77
3.6.5 Immunization and Infection of mice————————————————–78
3.6.6 T-Cell Proliferation Assay————————————————————–79
3.6.6.1 Cytokine Determination——————————————————–80
3.6.6.2 Nitrite Assay———————————————————————-81
3.6.7 Antibody Production———————————————————————-81
3.7 Statistical Analysis————————————————————————82
CHAPTER FOUR
4.0 RESULTS———————————————————————————–83
PLANT EXPERIMENTS
4.1 Extraction of plants————————————————————————83
4.1.1 Isolation of Compounds from Vernonia amygdalina——————————85
xiii
4.2 In Vitro Experiments———————————————————————-92
4.2.1 Macrophage Sensitivity Tests (Preliminary investigation)———————-92
4.2.2 Drug Sensitivity Assay on Macrophages——————————————–96
4.2.3 Cytotoxicity studies———————————————————————-100
4.2.4 Antitrypanosomal Activity————————————————————–102
4.3 In Vivo Antileishmanial Activity——————————————————-105
IMMUNOTHERAPY EXPERIMENT
4.4 Expression of Recombinant protein————————————————-110
4.5 Efficacy of DNA and Protein Vaccinations—————————————–113
4.6 Humoral Responses to DNA and Protein Vaccinations————————117
4.7 Cytokine and Nitric oxide Production———————————————–120
CHAPTER FIVE
5.0 DISCUSSION—————————————————————————–125
PLANT EXPERIMENTS
5.1 Plants Extraction————————————————————————-125
5.2 In Vitro Experiments——————————————————————–125
5.2.1 Macrophage Sensitivity Tests——————————————————–125
5.2.2 Cytotoxicity Tests————————————————————————128
5.2.3 Antitrypanosomal Activity————————————————————–131
5.3 In Vivo Experiment———————————————————————-132
IMMUNOTHERAPY EXPERIMENTS
5.4 Efficacy of DNA and Protein Vaccines———————————————134
5.5 Humoral Responses to DNA and Protein Vaccines—————————-136
5.6 Cytokine and Nitric oxide production in Vaccine groups———————-137
xiv
CHAPTER SIX
6.0 CONCLUSION AND RECOMMENDATIONS————————————141
6.1 Significant and original contribution to scientific knowledge——————143
References—————————————————————————————–144
Appendices—————————————————————————————–165
CHAPTER ONE
1.0 INTRODUCTION
Leishmaniasis is a parasitic disease caused by haemoflagellate protozoan of the
genus Leishmania. This protozoan was first described in 1903 by Leishman and
Donovan who worked separately (Herwaldt, 1999). These protozoan parasites
are members of the family Trypanosomatidae (order Kinetoplastida), which
comprises unicellular organisms, characterised by the presence of a single
flagellum and a DNA-rich mitochondria-like organelle called kinetoplast
(Descoteux and Turco 2002). It is a spectral disease with high clinical diversity,
which depends on the genetic potential, acute predisposition of the host’s immune
system (Kenner, 2007) and virulence of the leishmania specie involved as there
are over 20 species and at least 16 species are pathogenic for mammals (Acha
and Szyfres, 2003). It is transmitted by the bite of the female sandfly, which is a
small hairy mosquito-like insect. It is zoonotic as wild and domestic animals such
as dogs, rodents, foxes, wolves, jackals and other mammals serve as reservoir
hosts.
Leishmaniasis has been present in the Americas and Africa for several centuries.
It occurs from the tropical to Mediterranean regions and affects approximately 12
million people in 88 countries throughout Asia, Africa, Europe as well as North
and South America but mostly in developing countries. Also, over 350 million
people are at risk of infection in these areas. According to the World Health
organisation (WHO) there is an annual incidence of 1.5 to 2 million new cases.
The majority of cases are in the developing countries where approximately 90%
of all cases occur (WHO, 2005). The global incidence of this infectious disease
has increased in recent years because of increased international leisure,
2
missionary work and military-related travel, human alteration of vector habitats,
and concomitant factors that increase susceptibility, such as HIV infection and
malnutrition. In Africa, most cases are found in Sudan and Somalia while in
Nigeria, cases of cutaneous leismaniasis have been documented since the mid
20th century by a number of workers (McCulloch, 1930; Elmes and Hall, 1944;
Jelliffe, 1955; McMillan, 1957). It has been observed in Keana village in Awe,
Plateau state in the north central part of Nigeria (Agwale et al., 1993).
The clinical presentations are multifaceted but the leishmania species produce
three types of leishmaniasis, which are cutaneous, mucocutaneous and visceral.
Cutaneous leishmaniasis, caused by Leishmania major (L. major) is the focus of
this study. It is the most frequently occuring epidemics and is characterised by
clinical polymorphism (Masmoudi et al., 2007). It affects the skin, mucous
membrane or both tissues and is characterised by lesions, which start as small
red erythematous lesions at the site where the promastigotes are inoculated into
the skin by the bite of the sandfly. Microscopic examination of these lesions in
human tissue reveals the cytology of the parasites that present as amastigotes
which are ovoid or round shaped cells. These cells have relatively large nucleus,
thin cell membranes and rod-shaped kinetoplast. The amastigotes are found
within the macrophages, multiply in them and they serve as important models for
the understanding of the regulation of immune (Th cell) responses as well as the
immunomodulation of leishmaniasis (Alexander et al., 1999). Leishmaniasis
represents an emergent illness with high mortality and morbidity in the tropics and
subtropics (Dilvani et al., 2008). It is responsible for an estimated more than
80,000 deaths annually. Poor nutrition, infection, mass migration and
inaccessibility to regular healthcare in the developing world predispose people to
3
the disease and increase the mortality rate. The social burden of the disease is
also of great importance as it can cause severe deformities and disfigurements
which leads to social isolation (Renee, 2006). Treatment has proven to be difficult
due to the intramacrophagic location of the infectious forms, the amastigotes and
its control has largely been limited to the use of chemotherapeutic agents such as
the pentavalent antimonials. The efficacy of these antimonials has become
increasingly compromised due to the emergence of resistance within the parasite
population and the associated toxicity of the drugs. As a result of this, the search
for substances with antileishmanial activity, without toxic effects, and able to
overcome the emergence of drug resistant strains still remains the current goal
and research on this has intensified. This has led to the renewed interest in the
use of phytomedicine involving natural products and immunotherapy using
cytokines and vaccines. These have become areas of intense study in the search
for new treatments for this disease. The urgent need for alternative treatments
has led to a program to screen natural products for potential use in the therapy of
leishmaniasis and to develop a vaccine. Due to the limited availability of effective
pharmaceutical products, most people in areas where leishmaniasis is endemic
depend largely on popular treatments like cauterization procedures using copper
sulphate or battery acid and on traditional medical practices which depend heavily
on native plants to alleviate the symptoms (Carvalho and Ferreira, 2001) but
many of these have not been documented. Thus, as part of the search for new
drugs of high availability and less toxicity, the Tropical Disease Programme of the
World Health Organisation has considered the screening of these plants used in
tradomedicine for treatment of leishmaniasis as esential and of high priority. The
leads obtained from the screening of plants for antileishmanial activity resulted in
4
the discovery of Miltefosine, the first oral drug shown to be efficient and safe in
several clinical trials (Carvalho et al., 2000).
Nigeria, as in most cultures in the world has great potentials in terms of
biodiversity. In Nigeria, there are many local claims about medicinal plants and
treatment of common diseases (Raul et al., 1990). However, little is known about
indigenous plants with leishmanicidal activity hence, as part of the systematic
search programme for antileishmanial agents of plant-origin, the present study
assesses the leishmanicidal potentials of three Nigerian plants of medicinal value
used against other protozoan diseases. These are Azadirachta indica (neem
plant), Annona senegalensis (custard tree) and Vernonia amygdalina (bitter leaf).
Azadirachta indica is reputed to be of great medicinal value and products made
from it showed anthelmintic, antifungal, antidiabetic, antibacterial, antiviral and
anti-infertility activities and particularly prescribed for skin diseases (Puri, 1999).
Annona senegalensis has been used as an anti-trypanosomal (Igweh and
Onabanjo, 1989), anti-leishmanial (Sahpaz, et al., 1994) and antisplasmodic
(Langason et al., 1994). It is also used in the treatment of cancer (Graham et al.,
2000), malaria (Balansard and Timon-David, 1985), diarrhoea, dysentery (Kudi
and Myint, 1999), intestinal worms (Alawa et al., 2003), snakebites (Adzu et al.,
2005) and veneral diseases (Tabuti et al., 2003). V. amygdalina, is widely used in
Nigeria for human consumption, for ethnomedical practices like antiplasmodial,
anti-schistosomal (Jisaka et al., 1992a), anti-tumoral and anti-microbial actions
(Jisaka et al., 1993). It is also used for ethnoveterinary practices like anti-tick in
Ethiopia (Regassa, 2000) and as an anthelmintic in ruminants (Alawa et al.,
2003). V. amygdalina has been reported to be eaten by wild chimpanzees
possibly as self medication against endo-parasites (Huffman et al., 1993).
5
In the light of the above, the first part of this research work is intended to
determine any ethnopharmacological use of the three selected traditionally
administered plants in the north central part of Nigeria. These are plants
employed in the treatment of protozoan diseases such as trypanosomiasis and
malaria. Hence, we wish to extend use to another protozoan, Leishmania.
Ethnobotanical information and previous research works on these plants serve as
a basis for the selection of the plants, which are Azadirachta indica, Annona
senegalensis and Vernonia amygdalina. Thus, the aim of this study is to
determine if each of these plants possesses any leishmanicidal activity with the
following objectives:
To fractionate and purify the crude extract of the plants in order to
determine the constituent compounds
To investigate the in vitro effects of the crude extracts on the amastigote
form of L. major as well as the cytotoxicity of these plant extracts on
various cell lines i.e. normal (L929) and cancerous (Jurkat and SH-SY5Y)
To investigate the in vivo effects of the plant extracts and their effect on the
histopathology of the organs (skin, liver and spleen) in BALB/c mice
The aim of the second part of this work is to investigate the immunocompetence
of the DNA/Protein based vaccines with the following objective:
To determine if the DNA encoding GCS (pVAXGCS) and the
recombinant protein (rGCS) from L. donovani species used to immunize
BALB/c mice against L. major infection would confer cross protection as
well as stimulate any immunological responses in the mice.
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