ABSTRACT
Objective:
the study was designed to determine the anti-plasmodial and toxicological
effects of methanol extract of Chrysophyllum
albidium bark using albino male mice as models. Method: the antiplasmodial
effect and LD50 of methanol extract of Chrysophyllum albidium bark was determined in swiss albino mice
using standard methods. Blood samples were collected for the analysis of
selected biochemical parameters. Result: the LD50 of the methanolic
bark extract was estimated to be 750mg/kg body weight C. albidum methanolic bark extract (125-375mg/kg/day) showed
significantly (P<0.05) schizontocidal activities both in a 4day (early)
infection and in an established (7days) infection comparable to that of
chloriquine. The effect of oral administration of the bark extract of Chrysophyllum albidum did not show any
significant effect (P > 0.05) on the plasma concentration of total
bilurubin, albumin, total protein, alkaline phosphatase (ALP) as well as the
haemoglobin (Hb), red blood cell (RBC), mean corpuscular haemoglobin
concentration (MCHC), mean corpuscular volume (MCV) and packed cell volume
(PCV). The concentration of the platelet and white blood cell (WBC)
significantly decreased (P<0.05) at 125mg/kg body weight. The doses
significantly reduced (P<0.05) plasma levels of AST, ALT and creatinine.
Conclusion: C. albidum contains
anti-plasmodial substances which help to reduce parasitaemia and the results of
the biochemical and haematological parameters show that the extract is
non-toxic, this also validates the use of this plant as an antimalarial agent.
Recommendation: further studies needs to be done to identify and characterize
the active principles/substances in the extract.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of contents vi
List of tables vii
List of figures viii
Abstract ix
CHAPTER 1
INTRODUCTION
1.1 Background
of Study 1
1.2 Aim and Objectives 4
1.3 Justification of Study 4
CHAPTER
2
LITERATURE
REVIEW
2.1 Chrysophyllum
albidium (Africa star apple) 6
2.2 History
of Malaria 10
2.2.1 Origin
and prehistoric period 11
2.3 Pathophysiology
of Malarial Infection 13
2.3.1 Hemoglobin
degradation and the food vacuole 14
2.3.2 Proteases
and the food vacuole 15
2.4 Antioxidants 21
2.4.1 Metabolites 24
2.4.2 Relation
to diet 25
2.4.3 Pro-oxidant
activities 26
2.4.4 Enzyme
systems 26
2.4.5 Superoxide
dismutases (sods), catalase and peroxiredoxins 27
2.4.6 Thioredoxin
and glutathione systems 29
2.5 Phytochemicals
Role in Good Health 30
2.5.1 Role
of phytochemicals in disease conditions 31
2.6 Ethno
Medicinal Use of African Star Apple 36
2.6.1 Prevents
heart disease 36
2.9 Mechanism of Action of Chloroquine 37
CHAPTER 3
MATERIALS AND METHODS
3.1 Plant Collection,
Identification and Extraction 38
3.2 Laboratory Animals 39
3.3 Acute Toxicity Test and Determination of LD50 39
3.4 Parasite and
Infection 39
3.5 Experimental Design 39
3.6 Evaluation of Schizontocidal Activity in Early Infection
(4-day test) 40
3.7 Blood Sample Collection and Preservation 42
3.7.1 Hematological and
biochemical assays 42
3.8 Determination of
Serum Alanine Aminotransferase (ALT) Activity 45
3.8.1 Determination of serum
aspartate aminotransferase (AST) activity 46
3.8.2 Determination of serum
alkaline phosphatase (ALP) 47
3.8.3 Determination of serum
total protein 48
3.8.4 Determination of serum albumin 49
3.8.5 Determination of serum
creatinine 49
3.9 Phytochemical Screening 50
3.9.1 Determination of saponins 50
3.10 Determination of Flavonoids 51
3.11 Determination of Tannins 51
3.12 Determination of
Alkaloids 52
3.13 Determination of
Antioxidant Parameters 53
3.13.1 In-vitro determination
of 1, I-diphenyl-2-picrylhydrazyl 53
(DPPH)
radical scavenging activity of plant extract
3.13.2 Determination of nitric
oxide (No) radical scavenging activity 54
3.13.3 Determination of iron
chelating activity 54
3.13.4 Determination of
anti-lipid inhibition assay 55
3.14 Statistical Analysis 55
CHAPTER
4
RESULTS
AND DISCUSSION
4.1 Results 56
4.1.1
Parasitaemia results 56
4.1.1.1
Evaluation of schizontocidial activity in early infection (4-day test) 56
4.1.1.2 Evaluation of schizontocidal activity in
established infection 56
(curative
or rane test)
CHAPTER
5
DISCUSSION,
CONCLUSION AND RECOMMENDATION
5.1 Discussion
66
5.2 Conclusion 69
5.3 Recommendation
70
References 71
Appendixes
LIST OF TABLES
Table Title Page
4.1: Results
of hematological parameters 59
4.2: Results
of biochemical parameters 60
4.3:
Results of in-vitro
determination of DPPH radical 61
scavenging
activity of Chrosophyllum albidium
4.4: Results
of in-vitro determination of anti-lipid 62
4.5: Results
of in-vitro determination of nitric oxide 63
4.6: Results
of in-vitro determination of Iron chelating activity 64
4.7:
Results of phytochemical
screening of Chrysophyllum albidium 65
stem
bark
LIST OF FIGURES
Figure Title
Page
2.1:
Stem bark of African star
apple (udara) 6
2.2:
Fruits of African star
apple (udara) 6
2.3:
Leaves of African star
apple (udara) 7
2.4:
Ingestion of host
cytoplasm 15
2.5: Facilysin 17
2.6: Structure of heme 19
2.7: Activities and functions of the food vacuole 21
4.1 Results of schizonticidal
activity at day 4 57
4.4 Results of schizonticidal
activity at day 7 58
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND OF STUDY
Malaria remains one of the major killer diseases of
the world. It causes illness by the ability of the causative parasite to invade
the red blood cells (Beteck et al., 2014)
and the liver where they multiply. In extreme infections, up to 80% of the red
blood cells can be parasitized and destroyed (Katzung et al., 2012). This massive cell destruction is known to lead to
severe anaemia and clogging of the blood circulation of vital organs
particularly the brain and eventually death.
Antimalarias are agents used to inhibit the
development of plasmodium (the causative parasite of malaria) and they are
administered so they can completely destroy these parasites. There are quite a
wide range of antimalarias available. They may be classified according to the
different stages of the parasites they affect or according to their chemical
nature and function (Tripathi, 2009). All efforts made so far to eradicate
malaria using these agents and very highly effective residual insecticides
against mosquito have failed (Rang et
al., 2008). This is basically due to the increasing resistance of mosquito
to insecticides and the parasites to the drugs. One of the major challenges of
the medical world remains the eradication of malaria due to many reasons among
which is resistance to antiplasmodial agents. The need to achieve a more
radical cure of the malaria scourage remains with us considering the continuous
development of resistance to known and existing antimalarias being reported in
different parts of the world. Currently, the World Health Organization
guidelines for the treatment of malaria include the combination of one antimalaria
and one antibiotic provided that there is evidence of their efficacy and safety
(WHO, 2010). Combined therapy which will help in tackling this challenge has
therefore been recommended and efforts are one to find appropriate combinations
to be used. Combination therapy with antimalaria drug is the simultaneous use of
schizontocidal drugs with independent mode of action and different biochemical
targets in the parasites. These can either be fixed where the drugs to be
combined are co-formulated in the same tablet or capsule or non-fixed, where
they are co-administered in separate tablets or capsules. Drug combinations are
used to exploit the synergistic and additive potentials of each drug as well as
helping to improve efficacy while retarding the development of resistance to
individual components (Andrade et al., 2007).
One optimistic source for new affordable treatment against malaria lies in the
use of traditional herbal remedies. Despite the recent successes in rational
drug design and synthetic chemistry techniques by pharmaceutical companies,
natural products and particularly medicinal plants have remained an important
source of new drugs (Kaushik et al., 2013,
Lombardino and Lowe 2004). A definite virtue with medicinal plants is the rich
ethnopharmacological history of traditional knowledge and usage associated with
them. It is already providing a significant degree of protection to people at
large against malaria. However, if the gist of traditional knowledge can be
validated by scientific experiments, affordable and dependable cures can be
found against the drug resistant dreaded forms of malaria further, such
exploratory endeavours can pave the path for identifying novel pharmacophores
against malaria, which can be chemically synthesized and fine tuned as drugs of
the future.
As
a result of limited availability or affordability of pharmaceutical medicines
in many tropical African region has led majority remedies (WHO, 2002, Zirihi et al., 2005). Chrysophllum albidum (Linn), also known as African star apple
belongs to the family sapotaceae. It is widely distributed in the low land rain
forest zones and frequently found in villages (Madubuike and Ogbonnaya, 2003).
Across Nigeria, it is known by several local names and is generally regarded as
a plant with diverse ethno- medicinal uses (Amusa et al., 2003). In south- western Nigeria, the fruit is called
"agbalumo" and popularly referred to as "Udara" in
south-eastern Nigeria. It is a plant which has been used in traditional
medicine in Nigeria to treat health problem, phytochemical profile shows it
contains many biologically active substances that include alkaloids, tannin,
saponin etc (Okoli and Okere, 2010). Its rich sources of natural antioxidants
have been established to promote health by acting against oxidative stress
related disease such as diabetics, cancer and coronary heart disease (Bunts and
Bucar, 2002). The bark is used for the treatment of yellow fever and malaria
while the leaf is used as an emollient and for the treatment of skin eruption,
stomach ache and diarrhea (Adisa, 2000). Eleagnine, an alkaloid isolated from C. albidum seed. Cotyledon has been
reported to have anti-nociceptive, anti-inflammatory and anti-oxidant
activities (Idowu et al., 2006). This
research therefore is aimed at providing information on the possible anti-malaria
and toxic effects of the methanolic bark extract of C. albidum against plasmodium berghei
berghei infection of swiss abino mice.
1.2 AIM AND OBJECTIVES
The
aim of this study is to evaluate the anti-plasmodial and toxicological effects
of methanolic bark extract of chrysophyllum
albidum in albino mice.
Objectives
1.
Evaluation phytochemical
parameters
2.
Determination of lethal
dose of the extract (LD50)
3.
To determine the effect
of the extract on antioxidant parameters (in-vitro)
4.
Evaluation of
schizontocidal activity in early infection
5.
Evaluation of
schizontocidal activity in established infection
6.
Evaluation of the effect
of the extract on haematological parameters
7.
Evaluation of the effect
of the extract on serum parameters such as creatinine, AST, ALT, ALP,
Bilirubin, Total protein and Albumin.
1.3 JUSTIFICATION OF STUDY
In the absence of a credible vaccine and with
emergence of resistance to almost all anti-malaria drugs, the dream of
eradication of malaria appears to be a huge challenge, caused by a protozoan
parasite, malaria remains one of the dreaded diseases of the developing world
and Nigeria in particular, killing 367,000-755,000 people and causing disease
in 124-283 million people annually (WHO, 2014). The most severe manifestations
of malaria are caused by plasmodium falciparum. Even as malaria has been
affecting both the economic and Emotional aspects of mankind for a long time,
the relief against malaria has been coming in the form of herbal treatments,
such as cinchona bark and Quinghaosu plant
(leaves) which gave quinine and artemisnin respectively. The quinoline-based
quinine first and chloroquine later proved to be effective therapies against
malaria till resistance against quinolines began to surface and spread to large
parts of the world (CDC, 2012).
Against this scenario, artemisinin proved to be smart,
fast acting, potent drug against chloroquine-resistant malaria. However,
artemisinin resistance in the form of delayed clearance of the parasite is now
on the horizon (White, 2012). Conjuring images of a world where mankind may be
left with no effective drug against malaria. This calls for a rigorous search
for novel antimalarias and with this perspective in mind led to this research
work anti-plasmodial and toxicological effects of methanolic bark extract of Chrysophylium albidum using albino
mice.
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