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PROJECT TOPIC AND MATERIAL ON ANTIMALARIAL AND ANTIOXIDANT EFFECTS OF METHANOL AND FLAVONOID-RICH EXTRACTS OF Adansonia digitata STEM BARK ON Plasmodium berghei-INFECTED MICE

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ABSTRACT

Adansonia digitata has popular ethnomedicinal application in the treatment of malaria in
sub-Saharan Africa. The present study sought to investigate the antimalarial and antioxidant
effects of methanol and flavonoid-rich extracts of the stem bark on Plasmodium berghei-infected
mice in vivo. Thirty-five male mice, weighing 18-20 g and randomly allocated into seven groups
of five animals each were used. Group 1, which served as the positive control, was pretreated
with 1 ml/kg of the vehicle (5% v/v tween 80), mice in groups 2 and 3 were pretreated with 5
mg/kg b.wt of the standard drugs: chloroquine and arthemter/lumfantrine respectively, groups 4
and 5 were pretreated with 200mg/kg and 400 mg/kg methanol extract of A. digitata (ADME)
respectively while groups 6 and 7 were administered 200mg/kg and 400 mg/kg flavonoid-rich
extract of A. digitata (ADFE)respectively. Drugs were administered by oral gavage once daily
for five consecutive days before intraperitonial transfection of mice with an inoculum size of 1x
107of P.berghei. Blood was withdrawn from animals for the quantification of packed cell volume
(PCV) and parasitemia. Animals were anaesthetized with ether 72 h after transfection, dissected
and the livers quickly excised to prepare the homogenate used to evaluate the extent of
membrane lipid peroxidation and level of reduced glutathione (GSH).
ADME and ADFE treatment caused significant (P<0.001), dose-dependent
chemosuppresive activity and decreased parasitemia when compared with the infected, untreated
mice. Higher effective doses (400 mg/kg b.wt) of ADME and ADFE produced 68% and 82%
clearance of the parasites at day 5 after transfection compared with 100% clearance by both
standard drugs (chloroquine and arthemter/lumfantrine) at 5 mg/kg b.wt dosage. ADME and
ADFE at 400 mg/kg b.wt also reversed the malaria-dependent reduction in the PCV to preinfection
level and compared well with the reference drugs in this regard. ADME and ADFE at
iii
the evaluated concentrations significantly (P<0.001) reversed the elevated hepatic membrane
peroxidation caused by P. berghei infection but produced no significant effect on GSH when
compared with the infected, untreated mice.
The results of the present study revealed the antioxidant and prophylactic effects of
methanol and flavonoid-rich extracts of A. digitata on P. berghei-induced malaria in mice. It can
thus be concluded that the plant could be harnessed as source of antimalarial agents and further
justifies the folkloric use of the plant in the treatment of malaria.

TABLE OF CONTENTS

Title page i
Abstract ii
Certification: iv
Dedication v
Acknowledgement vi
Table of content viii
List of figures xii
List of tables xiii

CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Study Plant: Adansonia digitata 5
1.1.1 Ecology 9
1.1.2 Widespread use 9
1.1.3 Ethnomedicinal Use of A. digitata 10
1.2 Justification 12
1.3 Aim and Objectives of Study 12
1.3.1 Aim 12
1.3.2 Objectives 13

CHAPTER TWO
2.0 Literature review 14
2.1 Malaria 14
2.1.1 History 14
2.1.2 Prevalence 15
2.1.3 Causes of malaria 19
2.1.4 Malaria Vector 20
2.1.5 Malaria parasite and its life cycle 22
2.1.6 Symptoms of malaria 26
2.1.7 Diagnosis 27
2.1.8 Treatment 27
2.2 Oxidative stress in malaria 28
2.2.1 Host Immune Response as a Source of Oxidative Stress 30
2.2.2 Hemolysis as an Oxidative Stress Induction Factor in Malaria 31
2.2.3 Oxidative Stress and the Membrane of Infected-Erythrocyte 32
2.2.4 Oxidative Changes in Plasmodium 33
2.2.4.1 Production of Reactive Species by the Parasite 33
2.2.4.2 Antioxidant Defense Mechanisms in the Parasite 33
2.2.5 Antimalarial drugs and oxidative stress 34
2.3 Phytochemicals 36
2.3.1 Medicinal plants 37

CHAPTER THREE
3.0 MATERIALS AND METHOD 40
3.1 Materials 40
3.1.1 Chemical and Reagent 40
3.1.2 Plant material 40
3.1.3 Experimental animals 40
3.1.4 Parasite-Plasmodium berghei 41
3.2 Methods 41
3.2.1 Preparation of Crude Methanol Extract of Adansonia digitataStem Bark 41
3.2.2 Preparation of Flavonoid ExtractExtract of Adansonia digitata Stem Bark 41
3.2.3 Animal Treatment and Transfection 42
3.2.3.1 Grouping and Treatment of Experimental Animals 42
3.2.4 Quantification of Parasitemia and Packed Cell Volume Quantification 43
3.2.5 Sacrifice and Collection of Organs from Experimental Animals 43
3.2.6 Preparation of Tissue Homogenate 44
3.2.7 Estimation of Reduced Glutathione (GSH) Level 44
3.2.8 Assessment of Lipid Peroxidation 45

CHAPTER FOUR
4.0 Results 47
4.1 Antimalarial Effect of Methanolic and Flavonoid-Rich Extracts of A. digitata 47
4.2 Effect of Methanolic and Flavonoid-Rich Extracts of Adansonia digitata on
Pack Cell Volume of P. berghei-infected mice 49
4.3 Effect of Methanolic and Flavonoid-Rich Extracts of Adansonia digitata on
hepatic GSH level of P. berghei infected mice. 52
4.4 Effect of Methanolic and Flavonoid-Rich Extracts of Adansonia digitata on
hepatocytes membrane peroxidation in P. berghei infected mice. 52

CHAPTER FIVE
5.0 Discussion, Conclusion and Recommendation 57
5.1 Discussion 57
5.2 Conclusion 60
5.3 Recommendation 61

REFERENCES 62

LIST OF FIGURES
Figure 1.0: A. digitata tree plant 7
Figure 1.1: A. digitata leaves 8
Figure 2.1: Map showing the prevalence of Malaria in the world 18
Figure 2.2: Malaria vector, female anopheles mosquito 20
Figure 2.3: Life cycle of malaria parasite 25
Figure 2.4: Oxidative stress in malaria 29
Figure 4.1: Effect of methanol and flavonoid extracts of A. digitata on
hepatic GSH level of P. berghei infected mice 53
Figure 4.2: Effect of methanol and flavonoid extracts of A. digitata on
hepatocytes membrane peroxidation in P. berghei infected mice 55

LIST OF TABLES
Table 4.1: Prophylatic effects of methanolic and flavonoid-rich extracts of
A. digitata stem bark on P. berghei-infected mice 48
Table 4.2: Effects of methanolic and flavonoid-rich extracts of A. digitata stem bark
on packed cell volume P. berghei-infected mice 51

CHAPTER ONE

1.0 INTRODUCTION
Malaria is a mosquito-borne infectious disease of humans and other animals. It is a life threatening
blood disease caused by a Plasmodium parasite. The disease is transmitted most
commonly by an infected female Anopheles mosquito. The mosquito bite introduces the
parasites from the mosquito’s saliva into a person’s blood (WHO, 2014). The parasites travel to
the liver where they mature and reproduce. Five species of Plasmodium can infect and be spread
by humans. Most deaths are caused by P. falciparum because P. vivax, P. ovale, and P. malariae
generally cause a milder form of malaria. The species P. knowlesi rarely causes disease in
humans (Caraballo, 2014; WHO, 2014).
Malaria is the most important parasitic disease of man. This disease is presently endemic
and it is a major threat to public health in various parts of the world, around the equator and areas
such as parts of Asia, Latin America, Middle East, Eastern Europe, Pacific and much of Africa.
It is largely prevalent in these places and specifically accounts for 85-90% of fatalities in the Sub
Saharan Africa (Layne, 2007). The prevalence of malaria in the tropical and subtropical regions
have been attributed to rainfall, consistent high temperatures and high humidity as well as the
presence of stagnant waters in which mosquito larvae readily mature, thus providing a favourable
environment for the continuous breeding of this vector (Jamieson et al., 2006).
This disease is reasonably easy to recognize especially in patients with little or no
previous case(s) of malaria. The common symptoms include headache, fever, shivering, joint
pain, vomiting, hemolytic anemia, and jaundice, hemoglobin in the urine, convulsions and retinal
damage (Beare et al., 2006).
The signs and symptoms of malaria typically begin 8–25 days following infection
(Fairhurst and Wellems, 2010). It was however suggested that symptoms may occur later in
those who have taken antimalarial medications as prevention (Nadjm and Behrens, 2012).
Malaria can be diagnosed by the microscopic examination of a patient’s stained blood film. This
disease is often treated with antimalarial drugs depending on its type and severity.
Uncomplicated malaria may be treated with oral medications. The most effective treatment for
P. falciparum infection is the use of artemisinins in combination with other antimalarials (known
as artemisinin-combination therapy, or ACT), which decreases resistance to any single drug
component (Kokwaro, 2009).
Plant derived foods contain many bioactive compounds in addition to those which are
traditionally considered as nutrients, such as vitamins and minerals. These physiologically active
compounds, referred to simply as ‘phytochemicals’, are produced via secondary metabolism in
relatively small amounts (Rodriguez et al., 2006). Phytochemicals are chemical compounds that
occur naturally in plants (phyto means “plant” in Greek). They are non essential nutrients; some
are responsible for color and other organoleptic properties and may have biological significance
(US FDA, 2014).
The number of these phytochemicals has increased greatly over the last decades and
those of significant health benefits have been grouped into classes which include alkaloids,
terpenes, glycosides, flavonoids, phenolics, saponins, tannins, steroids etc. Phytochemicals
exhibit various pharmacological activities i.e. anti-inflammatory, antioxidant, anti-malaria, anticancer,
anaesthetics, and anti-viral, anti-fungal and anti-bacterial activities (Abdul Wadood et al.,
2013; Rodriguez et al., 2006).
Traditional medicines have been in use for the treatment of malaria for thousands of years
and are the source of the two main groups (artemisinin and quinine) of modern antimalarial
drugs (Kazembe et al., 2012). Medicinal plants contain some phytochemicals (bioactive
components) which exert definite physiological actions and are thus responsible for their
medicinal properties in curing diseases and herbal preparations account for 30-50% of total
medicine consumption (Abdul Wadood et al., 2013; Kazembe et al., 2012). The problems of
increasing pathogen resistance, e.g. Plasmodium to established antimalarial drugs (e.g. quinine,
chloroquine) coupled with the difficulties of the poor populace to afford and access effective
antimalarial drugs, have necessitated investigation of chemical compounds from plants for
antimalarial properties with the aim of finding novel drugs (Ibrahim et al., 2012). Recent
findings from various studies have boosted the confidence in the once abandoned herbs for the
treatment of resistant form of malaria parasites (Avwioro, 2010). This has the capacity to change
the perspectives on traditional medicine and its role in the health management, paving the way
for better collaboration between modern and traditional systems (Graz et al., 2011). Some of the
medicinal plants used for treating malaria include Artermisia annua (from which artemisinin was
obtained), Enantia chloranta, Carica papaya, Mangifera indica, Psidium guajava, Adansonia
digitata among others.
Adansonia digitata (baobab), a plant of the family of Bombaceae, is the most widespread
of the Adansonia species on the African continent, found in the hot, dry savannahs of sub-
Saharan Africa. In general, baobab is a good medicinal plant. Baobab pulp is rich in vitamin C,
the leaves are rich in good quality proteins – most essential amino acids are present in the leaves
and minerals, and the seeds in fat. Moreover, pulp and leaves exhibit antioxidant activity
(Chadare et al., 2009). A variety of chemicals have been isolated and characterized from A.
digitata. They belong to the classes of terpenoids, flavonoids, steroids, vitamins, amino acids,
carbohydrates and lipids (Donatien et al., 2011). Baobab bark is mainly used for medicinal
properties. The bark is thought to contain a bioactive component for treatment of malaria and
other fevers (Sidibe and Williams, 2002). Baobab bark which is often given to infants to promote
weight gain (Lockett and Grivetti, 2000) was found to be high in fat, calcium, copper, iron, and
zinc (Lockett et al., 2000).
A. digitata has a very high content of dietary antioxidant, including polyphenols, vitamin
C and E, carotenoids. This makes it effective in preventing oxidative stress related diseases
(Besco et al., 2007), such as inflammation, cardiovascular disease, cancer and aging related
disorders (Besco et al., 2007).
Evidences has also confirmed the use of the extracts of the leaves, fruits, seeds and bark
as an antimicrobial, antiviral (De Caluwe et al., 2010), anti-inflammatory and antipyretic drug
(Donatien et al., 2011). Powdered leaves are used as an anti-asthmatic and known to have
antihistamine and anti-tension properties (De Caluwe et al., 2010). Baobab bark is widely used
in traditional medicine as a substitute for quinine in case of fever or as a prophylactic (De
Caluwe et al., 2010).
Despite the large array of information about the therapeutical, neutraceutical,
cosmoteutical and ethnomedicinal uses of A. digitata, there is paucity of scientific information
about its antimalarial activity. The present research therefore sought to investigate the effect of
methanol and flavonoid-rich extracts of the leaf of A. digitata on the Plasmodium parasite.

1.1 Study Plant: Adansonia digitata
A. digitata (baobab tree in both English and French) is a characteristic plant of the Sahelian
region and belongs to the Bombaceae family (De Caluwé et al., 2010). The name commemorates
the French botanist Michel Adanson (1727- 1806). Linneaus dedicated the genus and species to
him; ‘digitata’ means hand shaped, referring to the shape of the leaf. Common names for the
baobab include dead-rat tree (from the appearance of the fruits), monkey-bread tree (the soft, dry
fruit is edible), upside-down tree (the sparse branches resemble roots) and cream of tartar tree
(cream of tartar). Baobab, a plant which derived its scientific name “A. digitata” from the French
explorer and botanist, Michel Adanson (1727-1806). He officially discovered it in 1749 on the
island of Sor in Senegal (Michel, 2015). “Digitata” refers to the digits of the hand. The Baobab’s
branches and leaves are akin to a hand. It is a traditional food plant in Africa that is high in
antioxidants, and has three times the vitamin C of an orange (The Independent, 2015).
The plant is a very massive tree with a very large trunk (up to 10 m diameter) which can
grow up to 25 m in height and may live for hundreds of years. It is widespread throughout the
hot and drier regions of tropical Africa (Donatien et al., 2011; De Caluwe et al., 2010).
A. digitata is a large, round canopied tree with a swollen trunk, about 10-25 m in height
(Gebauer et al., 2002), often with a bole of 3-10 m bark is soft, smooth, fibrous, reddish-brown,
greyish-brown or purplish-grey (Gebauer et al., 2002); bark of leaf-bearing branches is normally
ashy on the last node; a green layer below the outer, waxy layer of the bark, presumably to assist
in photosynthesis when the tree has shed its leaves.
The thick, fibrous bark is remarkably fire resistant, and even if the interior is completely
burnt out, the tree continues to live. Re-growth after fire results in a thickened, uneven
integument that gives the tree its gnarled appearance resembling an elephant’s skin but that
serves as added protection against fire. The fruit of the baobab tree hangs singly on long stalks
with an ovoid, woody and indehiscent shell 20 to 30 cm long and up to 10 cm in diameter (Nnam
and Obiakor, 2003), embedded in a whitish powdery pulp, have little or no endosperm. Leaves
alternate, digitately 3- to 9-foliate; leaflets oblong to ovate, 5-15 x 3-7 cm, lower leaflets being
the smallest and terminal leaflet the largest; leaflets dark green, with short, soft hairs; lateral
veins looping; apex and base tapering; margin entire; petiolules absent or almost so; petiole up to
12 cm long (Orwa et al., 2009). The ripe fruit pulp appears as naturally dehydrated, powdery,
whitish coloured and with a slightly acidulous taste (Vertuani et al., 2002).

Figure 1.0 A. digitata tree plant
Source: Wikipedia

Figure 1.1 A. digitata leaves
Source: Wikipedia

1.1.1 Ecology
The plant is widespread throughout the hot and drier regions of tropical Africa (Donatien
et al., 2011). The tree is characteristic of thorn woodlands of the African savannahs, which are
characterized by low altitudes with 4-10 dry months a year split into 1 or 2 periods. A. digitata is
resistant to fire, termite and drought, and prefers a high watertable. It occurs as isolated
individuals or grouped in clumps irrespective of soil type. It is not found in areas of deep sand,
presumably because it is unable to obtain sufficient anchorage and moisture. It is very sensitive
to water logging and frost. All A. digitata locations can be described as arid and semi-arid, with
not more than a day frost per year.

1.1.2 Widespread Use
Baobab tree has multi-purpose uses and every part of the plant is reported to be useful
(Donatien et al., 2011). Its leaves are used in the preparation of soup and they can also be
fermented and used as a flavouring agent, or roasted and eaten as snacks (Donatien et al., 2011).
The flower is eaten raw, the seeds also provide flour, which is very rich in vitamin B and protein,
and it is also used as baby food. The fruit pulp obtained from the seed provides a refreshing drink
when dissolve in water or milk (Donatien et al., 2011). The spongy and soft nature of the tree
makes it to store water, often chewed by human and animals during extreme scarcity of water.
The bark of the young baobab tree is used in making fishing nets, baskets, light canoes, trays,
mats and clothes (Rabi’u and Murtala, 2013; Tukur, 2010). The leaves of the baobab tree are a
staple food source for rural population in many parts of Africa especially the central part of the
continent (Gebauer et al., 2002; Tukur, 2010). Young leaves are widely used, cooked as spinach,
and frequently dried, often powdered and used for sources over porridges, thick gruels of grains
or boiled rice. The pulp serves as a fermenting agent in local brewing or as a substitute for tartar
in baking. The husk of the fruit is used in making dishes, vessels also as fuel. The roots also
provide a very important ingredient for dyes, the ash obtained from burning the tree is used in
soap making, and as fertilizer. The long-fibred wood is suitable for firewood. The shell and seeds
are also used for fuel, which potters use to smooth earthenware necklaces before firing. It is also
used for making gum or resin as glue can be made by mixing flower pollen with water.

1.1.3 Ethnomedicinal Uses of A. digitata
Various medicinal uses were discovered from the Baobab tree. The bark of the tree is
used in the treatment of fever; infections; wound disinfections; toothache etc. The leaves also are
used in the treatment of guinea worm sores, insect’s bites, kidney and bladder disorders,
diarrhea, ulcers, fatigue, cough, asthma etc (Donatien et al., 2011). The fruit pulp also provide
good medicine for malaria, small fox, dysentery and general fatigue for children while the seeds
are use in curing diseases like dental disorders. The roots of the tree (A. digitata) are used in the
treatment of malaria as well (Donatien et al., 2011).
Baobab fruit pulp has a well-documented antioxidant capability, a result of its high
natural vitamin C content (Blomhoff et al., 2010; Brady, 2011). Antioxidants could help prevent
oxidative stress related diseases such as cancer, aging, inflammation and cardio- vascular
diseases as they may eliminate free radicals which contribute to these chronic diseases
(Donatien et al., 2011; Blomhoff et al., 2010).
Baobab leaves, bark, pulp and seeds are used as food and for multiple medicinal purposes
in many parts of Africa (Diop et al., 2005). Baobab bark treats fever associated with illness
(Wickens and Lowe, 2008; Brady, 2011). Baobab fruit pulp has also been shown to lower
elevated body temperature without affecting normal body temperature (Donatien et al., 2011). It
is also used in cosmetic treatment; an infusion of roots is used in Zimbabwe to bathe babies to
promote smooth skin (De Caluwe et al., 2010). Seed oil is used to treat skin complaints (Sidibé
and Williams, 2002). Baobab fruit pulp has traditionally been used as an immunostimulant (Al-
Qarawi et al., 2003), anti-inflammatory, analgesic, antipyretic, febrifuge and astringent in the
treatment of diarrhoea and dysentery (Donatien et al., 2011) and to promote perspiration (Sidibe
and Williams, 2002).
The aqueous extract of baobab fruit pulp exhibited significant hepatoprotective activity
and, as a consequence, the consumption of the pulp may play an important part in human
resistance to liver damage in areas where baobab is consumed (Al-Qarawi et al., 2003). Oil
extracted from seeds is used for inflamed gums and to ease diseased teeth (Sidibe and Williams,
2002). Powdered leaves are used as a tonic and an anti-asthmatic and known to have
antihistamine and anti-tension properties. The leaves are also used to treat insect bites, Guinea
worm and internal pains, dysentery, diseases of the urinary tract, opthalmia and otitis (Sidibe and
Williams, 2002).
Baobab leaves are used medicinally as a diaphoretic, an astringent, an expectorant and as
a prophylactic against fever (Donatien et al., 2011). Leaves are used to treat kidney and bladder
diseases, asthma, general fatigue, diarrhoea, inflammations, insect bites and guinea worm
(Donatien et al., 2011). The widest use in tradition medicine comes from the baobab bark as a
substitute for quinine in case of fever or as a prophylactic. A decoction of the bark deteriorates
rapidly due to the mucilaginous substances present (Sidibe and Williams, 2002).
Moreover, the bark contains a white, semi-fluid gum that can be obtained from bark
wounds and is used for cleansing sores (Donatien et al., 2011). They contain the alkaloid
“adansonin”, which has a strophanthus-like action (Donatien et al., 2011).
In summary, A.digitata has been investigated for its anti-inflammatory properties (De
Caluwe et al., 2010), and its activity attributed to the presence of sterols, saponins and triterpenes
in the aqueous extract (Donatien et al., 2011; Brady, 2011). Its anti-pyretic activity (Donatien et
al., 2011), analgesic effect (Donatien et al., 2011; Masola et al., 2009), antimicrobial activity
(Yagoub, 2008), antioxidant property (Vertuani et al., 2002) has been confirmed and thus
attributed to the presence of various bioactive ingredients (Chadare et al., 2009).
1.2 Justification
The prevalence of malaria as well as the growing incidence of deaths resulting from the
disease coupled with the increase in the resistance of malaria parasite to synthetic drugs has led
to the increasing search for alternative treatment strategy. Plants constitute a natural reservoir of
phytochemicals with potentials for the treatment/management of many diseases. A. digitata has a
rich history of ethnobotanical usage in the treatment of a wide range of illnesses including
malaria but with paucity of scientific information in this regard. The present study is thus
necessary to fill this lacuna.

1.3 Aim and Objectives of Study

1.3.1 Aim
The aim of this study is to investigate the antimalarial activity of Adansonia digitata stem bark.

1.3.2 Objectives
The specific objectives of this study are to:
i. prepare the methanol and flavonoid-rich extracts of A. digitata stem bark;
ii. assess the antimalarial properties of the methanol and flavonoid-rich extracts of the stem
bark on plasmodium berghei-infected mice; and
iii. investigate the effect of the extracts on oxidative stress indices in Plasmodium bergheiinfected
mice.

 

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