Chapter One

Introduction

African animal trypanosomosis affects most domestic animals and is caused by blood dwelling

protozoan parasites of the genus Trypanosoma. Trypanosomosis is considered the most important

livestock disease after contagious bovine pleuropneumonia (CBPP) and remains a major obstacle

to livestock production in Nigeria. Trypanosomes are microscopic unicellular protozoa found in

the blood of man and some domestic and wild animals. They are also present in the saliva and

faeces of insect vectors. Transmission of Trypanosoma brucei between mammalian hosts is usually

by an insect vector, the tsetse fly. The parasite undergoes complex morphological changes as they

move between insect and mammalian hosts over the course of their variant surface glycoprotein

(VSG) coat, which undergoes remarkable antigenic variation, enabling persistent evasion of host

adaptive immunity and thus chronic infection (Assefa S. 2017).

1.1 Background of Study

Trypanosoma brucei species is the causative agent of Human African Trypanosomosis (HAT)

which is also known as sleeping sickness in man and Nagana or Animal African Trypanosomosis

(AAT) in animals. Trypanosoma brucei has traditionally been grouped into three subspecies;

Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense.

Trypanosoma brucei is a species of parasitic kinetoplastid belonging to the

genus Trypanosoma that is present in sub-Saharan Africa. Unlike other protozoan parasites that

normally infect blood and tissue cells, it is exclusively extracellular and inhabits the blood plasma

and body fluids. It causes deadly vector-borne diseases: African trypanosomiasis or sleeping

sickness in humans, and animal trypanosomiasis or nagana in cattle and horses. The first is a

1
parasite of non-human mammals and causes nagana, while the latter two are zoonotic infecting

both humans and animals and cause African trypanosomiasis (Assefa S. 2017).

Despite the many studies conducted on the control and prevention of trypanosomiasis, it is still

one of the limiting factors that greatly affects livestock and humans in sub-Saharan Africa (Assefa

S. 2017). Studies are in progress on developing vaccines against trypanosomiasis. However, the

use of chemotherapeutic agents is currently the existing method of treating the disease.

Trypanosomiasis weakens the immune system, which leads to inability of the hosts to fight

trypanosomes even after using trypanocides. In addition, the use of trypanocidal drugs, such as

diaminazene aceturate, Melarsoprol, Pentamidins, Fexinidazole and isomethamidium chloride,

etc. are faced with challenges, such as improper clinical efficiency, toxicity, and drug resistance

(Assefa S. 2017). These setbacks warrant a well-designed search for the development of potent

and not as toxic compounds from the available medicinal plants (Brady O, 2011).

Adansonia digitata which is also known as Baobab is a large iconic tree indigenous to Africa and

many other countries (Assefa S. 2017). It is considered as an emblematic, culturally important and

physically majestic sub-tropical tree. In the past decade, it has attracted the interest of several

pharmaceutical companies and researchers due to its various traditional uses, such as medicinal,

nutritional, and cosmetic. Various parts of the plant (e.g. leaf, bark, fruit pulp), have traditionally

been used as immunestimulant, anti-inflammatory, analgesic, insect repellent and pesticide. A.

digitata is being used in the treatment of diarrhea and dysentery in many African countries and has

been confirmed as a substitute for imported western drugs (Brady O, 2011). The high natural

content of vitamin C in Baobab fruit pulp is well-documented for its antioxidant capability, which

may be used in the prevention of oxidative stress and the treatment of related disease conditions

(Brady O, 2011).

2
1.2 Statement of the Problem

The impact of trypanosomosis on animal and human health and the economy is enormous thereby

necessitating continuous research for better ways of eradicating the disease. In order to tackle the

lingering problems associated with the chemotherapy of African trypanosomosis, in recent years,

scientists have focused their attention towards the search for effective ethno-botanical treatment

for the disease. Consequently, many African medicinal plants were discovered to have some

antitrypanosomal activities including A. digitata, and to the best of our knowledge there are no

reports on the oxidative effect of the extract of fruit pulp of A. digitata following trypanosome

infection.

1.3 Aim and Objectives of the Study

1.3.1 Aim

The aim of this study is to evaluate the anti-trypanasomal effects of Adansonia digitata (kuka) seed

extracts on Trypanosoma brucei brucei

1.3.2 Objectives

To identify the phytochemical constituents presents in the seed extract of Adansonia digitata

(kuka).

1.4 Significant of the Study

Nigeria’s biodiversity is rich in medicinal plants. Over 25% of our common medicines contain at

least some compounds obtained from plants (Farnsworth, 1988). The World Health Organization

reported that 70–90% of the worlds population relies chiefly on traditional medicine (WHO, 2004)

and a major part of the traditional therapies involve the use of plant extracts or their active

3
constituents. The local use of natural plants as primary health remedies is due to their

pharmacological properties. Many plant extracts owe their potency to the presence of metabolites.

These metabolites are usually found in various parts of the plants like roots, leaves, shoots and

bark. Many plants have therefore become sources of important drugs and as such the

pharmaceutical industries have exploited traditional medicine as a source of bioactive agents that

can be used in the preparation of synthetic medicine (Kinghorn, 1994). Natural products play

important roles in drug discovery and development process, particularly in the field of infectious

diseases, where 75% of these drugs are of natural origin (Newman et al., 2003).

4
 Chapter Two

Literature Review

Adansonia digitata, the African baobab, is the most widespread tree species of the

genus Adansonia, the baobabs, and is native to the African continent and the southern Arabian

Peninsula (Yemen, Oman). These are long-lived pachycauls; radiocarbon dating has shown some

individuals to be over 2,000 years old. They are typically found in dry, hot savannas of sub-

Saharan Africa, where they dominate the landscape and reveal the presence of a watercourse from

afar. They have traditionally been valued as sources of food, water, health remedies or places of

shelter and are a key food source for many animals. They are steeped in legend and superstition.

In recent years, many of the largest, oldest trees have died, for unknown reasons. Common names

for the baobab include monkey-bread tree, upside-down tree, and cream of tartar tree.

Scientific Classifications

Kingdom : Plantae

Clade: Tracheophytes

Claude: Angiosperms

Claude: Eudicots

Order: Rosids

Family: Malvales

Genus: Malvaceae

Species: Adansonia

Binomial Name

Adansonia Digitata L.

5
People have traditionally valued the trees as sources of food, water, health remedies or places of

shelter. The baobab is a traditional food plant in Africa, but is little-known elsewhere. Adanson

concluded that the baobab, of all the trees he studied, "is probably the most useful tree in all." He

consumed baobab juice twice a day while in Africa, and was convinced that it maintained his

health. According to a modern field guide, the juice can help cure diarrhoea.

The roots and fruits are edible. The fruit has been suggested to have the potential to improve

nutrition, boost food security, foster rural development and support sustainable land care. In

Sudan – where the tree is called tebeldi ‫ – يدلبت‬people make tabaldi juice by soaking and dissolving

the dry pulp of the fruit in water, locally known as gunguleiz. Water can also be extracted from

some of the trunks.

Baobab leaves can be eaten as a relish. Young fresh leaves are cooked in a sauce and sometimes

are dried and powdered. The powder is called lalo in Mali and sold in many village markets

in Western Africa. The leaves are used in the preparation of a soup termed miyan kuka in Northern

Nigeria and are rich in phytochemicals and minerals. The seeds can be pounded into a flour or to

extract oil for cooking. Baobab leaves are sometimes used as forage for ruminants in dry season.

The oil meal, which is a byproduct of oil extraction, can also be used as animal feed.

The fiber of the bark can be used to make cloth. In times of drought, elephants consume the juicy

wood beneath the bark of the baobab.

For export

In 2008, the European Union approved the use and consumption of baobab fruit. It is commonly

used as an ingredient in smoothies and cereal bars. In 2009, the United States Food and Drug

6
Administration granted generally recognized as safe status to baobab dried fruit pulp as a food

ingredient (Brady O, 2011).

2.2 Review of Past Related Work

Mohammad Madaki F et al., .Antioxidant and Anti-trypanosomal Activities of the Allium Sativum

(Garlic) Bulb Aqueous Extract on Trypanosoma Congolense Infected Mice. Iranian Journal of

Toxicology. 2022; 16(3):153-162. http://dx.doi.org/10.32598/IJT.16.3.746.1

In their study Trypanosomes cause the parasitic condition, which is transmitted by tsetse fly. The

disease is characterized by intermittent fever, anemia, and frequent diarrhea. This study examined

antioxidant and anti-trypanosomal effects of the aqueous extract of garlic in mice. The extract’s

phytochemical screening and antioxidant activity were performed based on standard methods. The

acute toxicity was evaluated via Lorke’s method and the antitrypanosomal effect was evaluated in

mice at 100, 250 and 500 mg/kg over 16 days. The screening identified phenols, flavonoids,

tannins, alkaloids and saponins. Phenols were present at the highest amount (291.88±6.12

mg/100g) and alkaloids were present the least (13.66±0.03 mg/100g). At 100 µg/mL, the extract

quenched 53.20% of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals with an inhibition

concentration (IC50) of 12.44 µg/mL. The lethal dose (LD50) of the extract was determined to be

>5000 mg/kg in mice. The extract exhibited high anti-trypanosomal activity at 500 mg/kg and

lowered the parasitemia count of 9.7±1.15. This was comparable to the diminazene aceturate

activity at 5 mg/kg. The extract at 500 mg/kg significantly increased the packed cell volume and

bodyweight of the infected mice. There were no significant differences in many hematological

indices comparing the control mice to those that received the extract at 500 mg/kg.The garlic

extract had a significant anti-trypanosomes effect and ameliorated the anemic condition induced

7
by infection with trypanosomes. Therefore, the extract may become a therapeutic candidate for the

management of trypanosomal infections.

Oluyomi Olajumoke Ogunleye, Isa Danladi Jatau, Audu Joseph Natala and Shola David Ola-

Fadunsin. Effects of aqueous extract of fruit pulp of Adansonia digitata L. on the oxidative stress

profile against Trypanosoma brucei brucei infection in albino rats. Ogunleye et al. Clinical

Phytoscience (2020) https://doi.org/10.1186/s40816-020-00203

In their study; Chemotherapy is the most widely used means of controlling trypanosomosis,

however, effectiveness of the drugs available is limited by a number of factors. This study

investigates the oxidative stress profile of aqueous extract of the fruit pulp of Adansonia digitata

on some organs in rats infected with Trypanosoma brucei brucei. Thirty-five male albino rats were

divided into 7 groups of 5 rats each. Groups B, C, D, E, F and G were inoculated with 0.20 ml of

suspension containing 106 T. b. brucei. Group A were neither infected nor treated. Group B were

infected but not treated. At onset of parasitaemia, rats in group C were treated with diminazene

aceturate at 3.5mg/kg body weight once, while rats in group D were treated with vitamin C at 200

mg/kg body weight for 3 days consecutively. Rats in groups E, F and G were treated orally for 3

days with the aqueous extract of fruit pulp of A. digitata at a dosage of 40 mg/kg, 80 mg/kg and

160 mg/kg body weight respectively. Liver and kidney tissues of the rats were collected at necropsy

(10 days PI) for oxidative stress analysis. There was a significant (p < 0.05) effect in the

concentration levels of malondialdehyde, superoxide dismutase, glutathione peroxidase and

catalase among the different groups treated with aqueous extract of fruit pulp of A. digitata. The

extract of A. digitata exert protective effects against tissue peroxidation in albino rats

experimentally infected with T. b. brucei.

8
Ogunleye, O.O et al.,Aqueous Extract Of Fruit Pulp Of Adansonia digitata (Linn): Phytochemical

Screening And In Vitro Antitrypanosomal Effect, Net Vet. J., March 2019 Vol 40 (1): 35 - 43.

https://dx.doi.org/10.4314/nvj.v40i1.3

In their study Chemotherapy is the most widely used means of controlling Trypanosomosis, a

major health problem to man and his livestock over much of Tropical Africa. However,

effectiveness of the drugs available is limited by a number of factors which include increasing

parasite resistance, treatment failures and unacceptable toxicity. This study investigated the

phytoconstituents of aqueous extract of fruit pulp of Adansonia digitata and its in vitro anti-

trypanosomal effects on Federe strain of Trypansoma brucei brucei. Qualitative phytochemical

analysis of the extract was carried out using standard technique. While in the in vitro study, about

3 ×105 T. brucei brucei in 0.3mls of blood suspended in 0.4mls Ringer’s solution were each

dispensed into tubes (A-D) containing 0.3mls of the aqueous extract at concentrations of

0.02mg/ml, 0.2mg/ml, 2mg/ml and 20mg/ml respectively. The fifth tube (E) was an untreated

control (Ringer’s solution and parasite). The tubes were incubated at 370C and examined for the

presence and motility of trypanosomes at 15 minutes intervals for 2hours. After the incubation and

motility assessment, 0.2ml of the contents of each tube was inoculated intraperitoneally into group

of 3 rats, 3 other rats served as uninfected controls. The inoculated animals were then examined

daily for the presence of trypanosomes for a period of 60 days. The phytochemical analysis showed

the presence of tannins, saponin, phenol, terpenoid, and cardiac active glycoside, anthraquinone,

reducing sugar, alkaloids, flavonoids and steroids. The extract demonstrated a concentration and

time dependent inhibitory effect on trypanosomal motility. Highest effect was observed at

concentration of 20mg/ml, with total ceassation of trypanosome motility from 75 minutes of

exposure all through the 120 minutes of the incubation. Also rats inoculated with content of the

9
tubes containing the 20mg/ml of the extract did not show parasitaemia and survived the 60 days

infectivity test period. However, all rats inoculated with trypanosomes exposed to lower

concentrations of the extract showed high parasitaemia with 100% mortality within 5 days post

inoculation.

Mergia et al., J Clin Exp Pathol Phytochemical Screening and In Vitro Antitrypanosomal Activity

of Aqueous and Methanol Leaf Extract of Verbascum sinaiticum (Scrophulariaceae) against

Trypanosoma congolense

In their study; Aqueous and methanol leaf extracts of V. sinaiticum were investigated for the

presence of secondary metabolites and their in vitro activity against Trypanosoma congolence, the

main causative agent of African animal trypanosomosis in Sub-Saharan Africa and Ethiopia. The

in vitro assay was carried out by monitoring test concentrations of 4, 2, 1, 0.4 and 0.2 mg/ml for

cessation or reduction in motility of trypanosomes followed by monitoring for loss of infectivity

to mice. Phytochemical screening revealed presence of alkaloids, flavonoids, glycosides, phenolic

compounds, saponins, steroids and tannins. An appreciable in vitro activity was attained by the

methanol extract of V. sinaiticum at 4 mg/ml concentration. In general, the results obtained suggest

ethnopharmacological usefulness of the plant and necessitate further studies to be carried on

isolated active substances from the plant.

10
 Chapter Three

Materials And Methodology

3.1 Study Area

3.1.1 Apparatus

1. Hammer

2. Mortar and pestle

3. Siever

4. Conical flask

5. Whatman filter paper No. 1

6. Weighing balance

7. Refrigerator

8. Cotton wool

9. Freeze-drying machine (ILSHIN freeze dryer with concentrator, Ilshin Lab. Co. Ltd,

Netherlands).

3.1.2 Reagents

1. Distilled water

2. HCL

3. Sulphuric acid

4. Dragendoff ‘s reagent

11
3.2 Experimental animals

The 10 albino mice was to be used in this study were obtained from the National Institute for

Trypanosomiasis and Onchocerciasis Research, Kaduna State, Nigeria. The mice, weighing

25.69±2.17g, were to be acclimatized for 6 days at the Science Laboratory Technology’s animal

holding unit, Department of Biochemistry, until the subsequent use for this research. All

experiments on the animals were performed using standard methods and in conformation with

established guidelines for the care and use of laboratory animals.

3.3 Collection and Preparation of Plant material

Dry fruits of Adansonia digitata will collected around the environs of Aliko Dangote University

of Science and Technology Wudil, Kano State, Nigeria. The fruits were cracked open using a

hammer, and the fruit pulp was manually harvested and allowed to further dry under room

temperatureon the laboratory bench and stored until use.

3.4 Preparation of Aqueous Metabolic Extract of Adansonia Digitata Seed

The fruit pulp was detached from the seed using mortar and pestle. The pulp was then separated

from the seeds and fiber by sieving. A total of 280 mg of the dried fruit pulp was weighed and

placed in a conical flask containing 7 litres of distilled water and allow to stand for 72 hours in a

refrigerator at +4°C with periodic agitation to ensure even mixture of the pulp with water. The

mixture was then filtered using 850nm and 150nm sieves in succession. The third stage of filtration

was done using Whatman Filter Paper No.1. Cotton wool was placed on the filter paper to facilitate

the filtration processes. It was then frozen and dried using freeze- drying machine (ILSHIN freeze

dryer with concentrator, Ilshin Lab. Co. Ltd, Netherlands).

12
3.5 Toxicity acute test

The extract was tested for acute toxic effect using method described by Lorke (1983). The test was

conducted in two phases. In phase one, nine rats weighing between 100 and 150 g were randomly

selected and used for the experiment. The nine rats were divided into three groups of three animals

each. Groups 1, 2 and 3 were given 10, 100, 1000 mg/kg of the aqueous extract respectively. All

the animals were observed for 24 hours for any sign of toxicity or death. In phase two of the trial,

which depended on the outcome of the first trial, three healthy rats were grouped into three

containing one animal each. Rats.

3.6 Parasite

Stabblate of Federe strain of Trypanosoma brucei brucei was obtained from the Nigerian Institute

for Trypanosomiasis Research (NITR), Kaduna and was maintained in the laboratory by passaged

into albino rats until when used.

3.5 Phytochemical screening of the extract

Phytochemical analysis of the aqueous extract of the fruit pulp of Adansonia digitata was carried

out according to the methods described by Sofowora (1993) and Evans (1998).

3.5.1 Test for Flavonoids

4mg/ml of each fraction of water and ethanol was added into a clean test tubes, and a piece of

Magnesium ribbon was added, then a drops of concentrated hydrochloric acid was also added. A

colour changed ranging from red crimson indicates a presence of flavonoids in the plant Materials

(Safowora, 1993).

13
3.1.2.2 Test for glycoside

Ten milliliters of 50% H2SO,4 was added to 1m each of water and ethanol filterates in separate

test tubes, and the mixtures was heated for 15 minutes, then 10ml of fehling’s reagents was added

And boiled. A brick red precipitates indicates the presence of glycosides in the plant materials,

(Safowora, 1993).

3.1.2.3 Test for Saponins

About 0.5g each of the powder plant materials of water and ethanol extracts was dispensed into

the test tubes, and 5ml distilled water was added and shaken vigorously. A persistent froth which

Lasts for 15 minutes indicates the presence of saponins (Brai and Turmer, 1975). Test for Tannins.

Two milliliters each of extracts materials were diluted with distilled water into the separate test

Tubes, 23 drops of 5% ferric chloride (Fecl) solution was added. A green black or blue Colouration

indicates the presence of tannins (Ciulci, 1994).

3.1.2.4 Test for Steroids

Two milliliters each of extract materials were evaporated to dryness in separate test tubes, the

Residues was dissolved in acetic acid anhydride, a few drops of chloroform was added, then a

Concentrated sulphuric acid were also added by using a dropping pipette via the side of the Test

tubes, formation of brown ring at the interface layers of the two liquids and violet colour in The

supernatant layers indicates the presence of steroids in the plant materials, (Ciulci, 1994).

14
3.1.2.5 Test for alkaloids

1m each of extracts fractions was added into a clean test tube, 2to 3 drops of dragendoff ‘s reagent

Was added. An orange red precipitates/turbidity was carefully observed, which indicates the

Presence of alkaloids compound in the plant materials (Ciulci, 1994).

15