OKONKWO, CHINELO HENRIETTA

(PG/M.PHARM/10/52905)

EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF

EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV

(APOCYNACEAE) IN MICE

Faculty of Pharmaceutical Sciences

Pharmacology And Toxicology

Chukwueloka.O. Uzowulu

Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF

EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV

(APOCYNACEAE) IN MICE

BY

OKONKWO, CHINELO HENRIETTA

(PG/M.PHARM/10/52905)

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

SEPTEMBER, 2015

EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF

EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV

(APOCYNACEAE) IN MICE

BY

OKONKWO, CHINELO HENRIETTA

(PG/M.PHARM/10/52905)

A PROJECT REPORT PRESENTED TO

THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF

MASTER OF PHARMACY

(M.PHARM) DEGREE

DR ADAOBI C. EZIKE (SUPERVISOR)

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

SEPTEMBER, 2015

TITLE PAGE

EVALUATION OF THE ANTIPLASMODIAL ACTIVITY OF METHANOL LEAF

EXTRACT AND FRACTIONS OF LANDOLPHIA OWARIENSIS P. BEAUV

(APOCYNACEAE) IN MICE

CERTIFICATION

OKONKWO, CHINELO HENRIETTA, a postgraduate student of the Department of

Pharmacology and Toxicology with Registration Number PG/M.PHARM/10/52905, has

satisfactorily completed the requirements for the award of the degree of Master of Pharmacy

(M.Pharm) of the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical

Sciences, University of Nigeria, Nsukka.

The work embodied in this project is original and has not been submitted in part or full for

any other diploma or degree of this or any university.

…………………………… ……………………………

DR ADAOBI C. EZIKE DR ADAOBI C. EZIKE

Supervisor Head of Department

DEDICATION

This work is dedicated to God Almighty “for nothing is impossible with him” (Luke 1:37)

and also to my husband and three children as well as my parents.

ACKNOWLEDGEMENTS

I express my profound gratitude to God who out of His abundant love saw me throughout my

education in this university and my research into this project “for in Him we live and move

and have our being” (Acts 17: 28).

I am sincerely grateful to my supervisor Dr Adaobi C. Ezike for her motherly love, untiring

suggestions, constructive and objective criticisms which have led to the successful

completion of this work.

With heartfelt gratitude, I esteem highly my husband, Emmanuel and children Chisom,

Chibuikem and Chidubem as well as my parents Mr & Mrs G.O. Ojielo, my brothers and

sisters who are Okey, Maureen, Chioma, Chibueze, Chinedu, Nneka and Chinwe for their

love, understanding, patience, encouragement, prayers, financial and moral support.

My special thanks to Pharm Collins Onyeto and Prof. M. Emeje of NIPRD, for their prayers,

encouragement and relentless effort in making this work a reality. I also appreciate Mr Austin

Okorie for all his efforts and assistance in the laboratory, Mr Alfred Ozioko and Dr Michelle

of InterCEDD, Rev Ezea of Pharmacognosy as well as Dr B. Adzu, Dr S. Ameh and Mrs

Gloria of National Institute of Pharmaceutical Research and Development (NIPRD) for their

various contributions to the successful completion of this work.

I am particularly grateful to all my lecturers in the department especially Prof A Akah, my

pharmacology father, Prof Charles Okoli, Dr C.S. Nworu, Dr T.C. Okoye, for the vast

knowledge I acquired from them which has contributed towards the success of this work as

well. I am also grateful to my friends Pharm (Mrs.) F Mbaoji, Pharm (Mrs) Ifeoma and the

entire staff of the Department of Pharmacology and Toxicology especially Mr Bonny Ezeh,

Mrs Ego and Mrs Ugwu for their assistance and encouragement.

I am also thankful to my former Head of Department at National Hospital Abuja, Pharm

Abdu Msheliza and his family and also Pharm C.A. Umezulike, Pharm (Mrs) A. Ajemigbitse,

Dr A. Akinmola, Dr Ogunfowokan, Dr Orilade, Allison all of National Hospital Abuja for

their understanding, suggestions and encouragement in seeing to the successful completion of

this work.

I register my gratitude too to all my friends in the university as well as classmates in the Dept

of Pharmacology and Toxicology among who are Ben, Dr Ugwu, Dr Chibueze, Ufere,

Chinenye and all others not mentioned for their encouragement in their various capacities to

my success.

I appreciate Revds Sam Etim (MSP), Iffiok E Inyang (MSP), Festus Ejiofor (MSP), Srs Cecilia

Azuh, Lucy and Evarista (MMM) for their encouragement, moral and spiritual support given

to me marvelously in different ways.

I equally appreciate all my friends namely: Lucy, Ben, Chinelo, Juliet Okonkwo, Ewi, Ohi,

Immaculata, Dr Ogunleye and many others not mentioned here for their various contributions

and support.

Finally, I am sincerely grateful to all the staff of NIPRD, Idu, Abuja, for their various

assistance and granting me access to their laboratory and also Dr Aina of National Institute of

Medical Research (NIMR), Lagos for providing the parasite used for this work.

ABSTRACT

Landolphia owariensis P. Beauv (Apocynaceae) is a woody liane commonly used in Africa

for the treatment of gonorrhea, worm infestation and malaria. The methanol leaf extract (ME)

of L. owariensis was obtained by cold maceration and then fractionated into nhexane (nHF),

ethylacetate (EF) and methanol (MF) fractions. The methanol extract and fractions were

tested against chloroquine-sensitive Plasmodium berghei berghei in early, established and

repository models of infection using Peter’s 4-day suppressive model, Rane’s curative model

and Peters prophylactic model respectively. The antiplasmodial activity was evaluated by

determining the parasitemia, body weight and survival time of each of the eighty-four mice

comprising six mice per group. Groups 1-12 were given graded doses of 200, 400 and 800

mg/kg body weight of extract or fractions respectively while group 13 and 14 received 5

mg/kg/day of chloroquine and 3% Tween 80 respectively. All administration was orally.

Acute toxicity was studied using modified Lorke’s method. Phytochemistry of extract and

fractions as well as HPLC fingerprinting of ME, EF and MF were also carried out. The

methanol extract and all the fractions exhibited significant (P<0.05) but varying levels of

antiplasmodial activity comparable to the group treated with chloroquine. MF elicited the

highest chemosuppression of 96.04% at 800 mg/kg with the prophylactic model while nHF

elicited the least activity with chemosuppression of 29.38-58.75% at 200 -800 mg/kg

respectively. The phytochemical screening of the extract and fractions revealed the presence

of secondary metabolites. The LD50 was estimated to be greater than 5000 mg/kg p.o in mice.

HPLC analysis of ME, EF and MF showed different peaks representing different

components. The results of this study suggest that the leaf extract and fractions pose

significant antiplasmodial activity.

TABLE OF CONTENT

Page Title page …...................................................................................................................... ii

Certification ………………………………………………………………..................... iii

Dedication ……………………………………………………………………………… iv

Acknowledgement ………………………………………………………....................... v

Abstract …………………………………………………………...……….………….. vii

Table of content ............................................................................................................. viii

List of tables ………………………………………………………………................... xii

List of figures …………………………………………………………………………. xiii

CHAPTER ONE: INTRODUCTION

1.0 Introduction ………………………………………………………………………... 1

1.1 Background ……………………………..……...…….…………………………….. 1

1.2 Definition of Malaria ……………………..………….…………………………….. 2

1.3 Epidemiology of Malaria …..….…………………………………………………… 2

1.4 Etiology of Malaria .............................................................................................…... 3

1.4.1 Vector ……………………………………………………………………………… 4

1.4.2 Biology of Malaria Infection (Life cycle of Malaria Parasite) .……………….…... 5

1.4.3 Pathogenesis of Malaria ...................................................................................…... 8

1.5 Clinical Manifestation of Malaria ...…………..………...………………………….. 9

1.6 Diagnosis of Malaria ………………..………………………….…………………. 10

1.7 Classification of antimalarials ………………………….......................................... 11

1.8 Chemotherapy of Malaria .................................................................................…... 13

1.8.1 Chloroquine …………………………………………………………………....... 14

1.8.2 Amodiaquine …………………...……………………………………………… 16

1.8.3 Quinine and quinidine ............................................................................…........... 16

1.8.4 Mefloquine .…………………………………………….……………………...... 17

1.8.5 Halofantrine .……………………………………………………………………. 17

1.8.6 Lumefantrine ..................................................................................................…... 18

1.8.7 Primaquine ……………………………………………..………………………. 19

1.8.8 Sulphadoxine - Pyrimethamine ......................................................................…... 19

1.8.9 Antibacterial agents ......................................................................................….... 20

1.8.10 Artemisinins …………………………………….……………………............... 20

1.8.10.1 Antimalarial Properties of Artemisinins .....…………………………………... 21

1.8.10.2 Artemisinin Based Combination Therapy (ACT) …………………………….. 22

1.9 Prevention and Control of Malaria ……………………………………………….. 24

1.10 Review of Plants with Antimalarial Activity …………………………………….. 26

1.11 Botanical Profile of Landolphia owariensis ……………………………………… 30

1.11.1 Plant Taxonomy ………………………………………………………………… 30

1.11.2 Description of Plant …………………………………………………………….. 31

1.11.3 Geographical Distribution of Plant .…………………………………………….. 31

1.11.4 Ecology …………………………………………………………………………. 32

1.11.5 Ethnomedicinal Uses …………………………………………………………….33

1.12 Literature Review ………………………………………………………………… 33

1.13 Aim …………………….…………………………………………………………. 34

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials ...............................................................................................…............... 35

2.1.1 Animals …………………………….…………...................................................... 35

2.1.2 Drugs …………………………………………………………………………… 35

2.1.3 Chemicals and Reagents ……………………………………………………….. .35

2.1.4 Equipment ......................................................................................................…... 36

2.2 Methods …………………………………………………….................................... 36

2.2.1 Collection, Authentication and Preparation of Plant Materials ………………….. 36

2.2.2 Preparation of Crude Extract …………………………………………………….. 37

2.2.3 Solvent-guided Fractionation of Crude Extract ………………………………….. 37

2.2.4 Determination of Yield (%) ……………………………………………………… 37

2.2.5 Phytochemical Analysis of Extract and Fractions ……………………………….. 38

2.2.5.1 Test for Saponins ………………………………………………………………. 38

2.2.5.2 Test for Tannins ….…………………………………………………………….. 38

2.2.5.3 Test for Flavonoids …………………………………………………………….. 38

2.2.5.4 Test for Resins …………………………………………………………………. 39

2.2.5.5 Test for Steroids and Terpenoids ………………………………………………. 39

2.2.5.6 Test for Alkaloids ……………………………………………………………… 39

2.2.5.7 Test for Glycosides …………………………………………………………….. 40

2.2.5.8 Test for Fats and Oils ………………………………………………………….. 40

2.2.5.9 Test for Carbohydrates ………………………………………………………… 40

2.2.5.10 Test for Reducing Sugar ……………………………………………………… 40

2.2.6 High Performance Liquid Chromatography (HPLC) Analysis ………………….. 41

2.3 Pharmacological Studies …………………………………………………………… 42

2.3.1 Determination of Acute Toxicity (LD50) of ME …………………………………. 42

2.3.2 Rodent Parasite (Plasmodium berghei berghei NK65) ………………………….. 43

2.3.3 Parasite Innoculaton ……………………………………………………………… 43

2.3.4 Evaluation of Activity on Early Malarial Infection (4-Day Suppressive Test) ….. 43

2.3.5 Evaluation of Activity on Established Infection (Curative or Rane Test) ………. 45

2.3.6 Evaluation of Prophylactic Activity (Repository Test) ………………………….. 46

2.4 Statistical Analysis …………………………………………………………………. 46

CHAPTER THREE: RESULTS

3.1 Extraction and Fractionation ……………………………………………………….. 47

3.2 Phytochemical Analysis of extract and fractions …………………………………... 47

3.3 High Performance Liquid Chromatography (HPLC) Analysis ……………………. 50

3.4 Acute Toxicity (LD50) of ME ……………………………………………………… 50

3.5 Pharmacological Studies …………………………………………………………… 55

3.5.1 Effect of ME and Fractions on Early Malaria Infection (4-Day Suppressive Test) 55

3.5.2 Effect of ME and Fractions on Established Infection (Rane Test) ………………. 57

3.5.3 Prophylactic Effects of ME and Fractions against P. berghei berghei infected mice

…….…………………………………………………………………………………….. 60

CHAPTER FOUR: DISCUSSION AND CONCLUSION

4.1 Discussion ………………………………………………………………………….. 62

4.2 Conclusion …………………………………………………………………………. 66

References ……………………………………………………………………………… 67

LIST OF TABLES

Table 1 List of some plants with antimalarial activity ……………................................. 29

Table 2 Yield (%) of extract and fractions ……………………………………………... 48

Table 3 Phytochemical constituents of extract and fractions ……………………........... 49

Table 4 Acute toxicity testing of methanol leaf extract and fractions of Landolphia

owariensis in mice ……………………………………………………………………... 54

Table 5 Effects of methanol extract and fractions of Landolphia owariensis on early infection

against Plasmodium berghei berghei infected mice

…………………………………………………………………...................................... 56

Table 6 Parasitemia measurement of methanol extract and fractions of Landolphia owariensis

on established infection

...……………………….………………………………………………………………... 58

Table 7 Effect of methanol extract and fractions of Landolphia owariensis on established

infection against Plasmodium berghei berghei infected mice

……………………………………...…………………………………………………… 59

Table 8 Effects of methanol extract and fractions on prophylactic infection against

Plasmodium berghei berghei infected mice

…………………………………………………………………………........................... 61

LIST OF FIGURES

Figure 1: Life Cycle of Plasmodium ……………………………………………………. 7

Figure 2: L. owariensis in its natural habitat …………………………………………… 32

Figure 3: HPLC fingerprint profile of methanol extract of L. owariensis

…………….………………………………...................................................................... 51

Figure 4: HPLC fingerprint profile of methanol fraction of L. owariensis

………………………………………………………..…………………………………. 52

Figure 5: HPLC fingerprint profile of ethylacetate fraction of L. owariensis

..…………………………………………………………...…………………………….. 53

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background

Malaria presents a global devastating burden. The latest estimates released December 2013

indicate that about 207 million cases of malaria were reported in 2012 with an uncertainty

range of 135 million to 287 million and an estimated 627 000 deaths with an uncertainty

range of 473 000 to 789 000 (WHO, 2014). A child dies every minute from malaria in Africa.

Malaria though a complex disease is preventable and curable (WHO, 2008a; WHO, 2014). It

is a disease caused by infection with single-celled protozoan parasites of the genus

Plasmodium.

There has been a dramatic increase in both international and domestic funding for malaria

control since the last decade as well as an increasing national political commitment to

controlling malaria and intensified efforts in endemic countries (WHO, 2008b; Pigott et al.,

2012). Malaria control has also featured high on the world’s health and development agenda

since the launch of Roll Back Malaria Initiative by World Health Organization (WHO) in

1998. The inspiration behind this initiative was to alleviate poverty and strengthen health

systems in malaria endemic countries in such a way that the enormous public health problem

caused by malaria will be fundamentally addressed (WHO, 2008b). Despite the intensified

efforts and initiative, eradication of malaria still poses a global challenge and threat. Malaria

occurs in 109 countries of the world in which Nigeria is one of the 5 main contributors of

50% of global death and 47% of malaria cases. Global strategy consists of three components

that will ensure reduction in mortality and morbidity of malaria. This includes control with

existing tools, elimination by complete interruption of malaria transmission and continued

research to create new tools and approaches (WHO, 2008a).

1.2 Definition of Malaria

Malaria has variety of meanings but can be defined in general terms as a parasitic disease,

illness, infection or a public health problem caused by protozoa of the genus Plasmodium

which afflicts an individual or a community (Koram and Molyneux, 2007). Malaria is an

acute parasitic disease due to Plasmodium spp infection of the red blood cells leading to

constellation of signs and symptoms such as cyclical fever, muscle pains, headache,

vomiting, diarrhoea, cough etc. which if untreated can rapidly lead to complications and

death.

1.3 Epidemiology of Malaria

The distribution and intensity of malaria is geographically specific. Even though malaria is

prevalent in poor countries, its severity and difficulty in controlling it is primarily determined

by climate and ecology. Other determinants include the general level of urbanization like

inadequate sewage disposal and treatment, poor hygiene practices, lack of safe drinking water

and substandard housing conditions which are all direct consequences of poverty. Personal

behavior such as inability to carry out outdoor spraying or use insecticide treated nets also

contribute to the poor difficulty in controlling malaria (Gallup and Sachs, 2001).

Epidemiology of malaria is driven by the goal of malaria control and elimination which is to

reduce morbidity and mortality of malaria as well as interrupt the chain of local transmission.

Therefore, understanding the dynamics of malarial transmission and vectorial capacity is

fundamental to achieving this goal (Gallup and Sachs, 2001; WHO, 2008b).

In Sub Saharan African countries where data collection systems are often rudimentary,

accurate statistics due to malaria deaths are rarely available and childhood fever or other

illnesses are majorly attributed to malaria, (Iley, 2006). Despite the high mortality rates,

existing vital registration systems are not reliable or comprehensive and most deaths occur

outside the health facilities (Snow and Omumbo, 2006). Invariably, there is lack of precision

in current malaria statistics (Snow et al., 2005; Bell et al., 2005). This presents an interesting

epidemiological challenge to sub Saharan Africa. Sequel to this is the documented evidences

on overdiagnosis and misdiagnosis of malaria where malaria cases are reported and treated

despite laboratory confirmation of no parastemia (Reyburn et al., 2004; Gwer et al., 2007;

Nankabirwa et al., 2009; Leslie et al., 2012; Oladosu and Oyibo, 2013).

In Nigeria however, malaria constitutes an economic burden with an annual economic loss of

about 132 billion Naira as, cost of treatment, prevention, loss of man-hour among others per

annuum and also accounts for 60% outpatient visits as well as 30% hospital admissions.

1.4 Etiology of Malaria

There are five species of Plasmodium known to infect humans, Plasmodium falciparum,

Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi

(Vinetz et al., 2011). The four identified species of this parasite causing human malaria are P.

falciparum, P. vivax, P. ovale and P.malariae Out of these four known parasites, P.

falciparum accounts for 98% of all cases of malaria in Nigeria (Onwujekwe et al., 2000;

Federal Ministry of Health, 2005; Jimoh et al., 2007). This is the species that is responsible

for the severe form of the disease that is potentially life threatening with a documented

estimate of 300,000 deaths each year (Federal Ministry of Health, 2009; National Population

Commission, 2008).

Most of the malarial infections are caused by P. falciparum and P. ovale. Infections resulting

from P. falciparum are not only associated with majority of the burden of malaria in sub –

saharan Africa but found to be associated with high level of resistance (Mendis et al., 2001;

Vinetz et al., 2011). P. knowlesi which has now emerged as a zoonotic malarial parasite can

cause lethal human malarial infection in some parts of South East Asia like Malaysia,

Indonesia, Thailand, Singapore and the Philippines. It was initially thought to infect only

non-human primates (Cox-Singh et al., 2008).

1.4.1 Vector

Mosquitoes are small, long legged, two winged insects belonging to the order Diptera, and

family Culicidae. They form a monophyletic assemblage that is currently divided into forty-

four genera and over 3,500 species described world-wide. It is the female mosquito of the

genus Anopheles that is the vector responsible for the transmission of malaria parasites to

humans. Mosquitoes can further be divided into two groups according to their reproductive

strategy. Autogenous mosquitoes which produce their first batch of eggs without a blood

meal relying solely on nutrients acquired during their larval stages but must blood feed to

produce subsequent batches of eggs. In contrast to this, anautogenous mosquitoes require

vertebrate blood meal for egg production. Male mosquitoes do not lay eggs hence do not

require blood meal and invariably do not bite (Gulia-Nuss et al., 2012; Hansen et al., 2014).

This vector carrying plasmodium predominantly feed indoors upon humans unlike outdoors

where host availability is haphazard causing the insect to lose energy during host searching.

The indoor (endophagic) feeding behaviours enhance mosquitoes survival as biting is done

preferentially at night when the host is available, stable and sleeping (Killeen et al., 2013).

1.4.2 Biology of Malaria Infection (Life Cycle of Malaria Parasite)

Understanding the lifecycle of the malaria parasite is a prerequisite to tailoring drug therapies

to the various species and geographical contexts. Malaria infection is initiated when

Plasmodium sporozoites are inoculated into the bloodstream of humans following the bite of

a Plasmodium infected female anopheles mosquito. The sporozoites travel to the liver within

minutes to infect the hepatocytes via cell surface receptor-mediated events and initiate the

asymptomatic prepatent period also known as exoerythrocytic stage of infection (Vinetz et

al., 2011). During this period which lasts for about one week, asexual replication occurs

leading to the production of liver stage schizonts. This eventually ruptures to release tens of

thousands of merozoites into the blood stream to infect the red blood cells. This initiates the

asexual erythrocytic stage of malaria parasite which is responsible for the clinical

manifestation of malaria. Mechanisms of red blood cells invasion is through merozoite

recognition of the red blood cells, mediated by their binding to specific cell surface receptors.

Majority of the merozoites develop into ring form inside a red blood cell which becomes

trophozoites that mature into an asexually dividing blood stage schizont while a small

proportion become gametophytes, the form of the parasite that is infective to mosquitoes.

When the infected erythrocytes rupture, the schizonts release 8-32 merozoites that can

establish new infections into nearby red cells. The duration of erythrocyte replication cycle

varies for different species and this includes 24 hours for P. knowlesi, 48 hours for P.

falciparum, P. vivax and P. ovale while P.malariae lasts 72 hours (Vinetz et al., 2011) hence

tertian and quartan malaria. The life cycle of malaria parasite in the vector begins when an

uninfected mosquito ingests gametocytes into its midgut during an infectious blood meal

which transform into gametes that can fertilize to become zygotes. Ookinetes formed at the

maturation of the zygotes penetrate the mosquito midgut and further develop into oocytes

where numerous rounds of asexual replication occur over 10 – 14 days to generate sporzoites.

It is the fully developed sporozoites that rupture from the oocytes to invade the mosquito

salivary glands from which they initiate a new infection during any blood meal (Vinetz et al.,

2011). The complete life cycle of malaria parasite in the different hosts is illustrated in Figure

1.

Figure 1: Life Cycle of Plasmodium

1.4.3 Pathogenesis of malaria

The pathogenesis of human malaria is caused by intra-erythrocytic growth of the parasite.

This is apparent when each sporozoite releases about 30,000 merozoites to infect the red

blood cells after the exoerythrocytic stage of malaria infection (Garcia et al., 2001). The entry

of the parasite into an erythrocyte triggers its metamorphosis to a complex rich in

endomembrane system and complex trafficking events. From the biology of the erythrocytic

phase of P. falciparum, endothelial adherence or vascular sequestration of parasitized red

cells occurs inevitably before the onset of illness. This sequestration lasts half of the 48 hour

erythrocytic cycle (Pavithra et al., 2004). Fever results from the rupture of infected

erythrocytes with the release of schizonts which can infect new cells. A striking characteristic

of this fever is the periodicity of its occurrence depending on the species of the malaria

parasite. Fever and chills generally occur every 3rd day (tertian periodicity) in P. falciparum,

every 2 days (tertian periodicity) in P. vivax and every 4 days (quartan periodicity) in P.

malaria (Garcia et al., 2001).

The pathogenesis of malaria is complex due to the fact that it entails immunologic and non-

immunologic mechanisms. For instance, there is a dysfunction arising from alteration of

many tissues and organs in severe malaria which consequently leads to metabolic acidosis

and ischemia. Also, the ability of the parasite to damage the infected red blood cells and

induce production of pro-inflammatory cytokines consequently results to severity of the

disease (Angulo and Fresno, 2002). The release of the pro-inflammatory cytokine tumor

necrotic factor (TNF) is hypothesized to be responsible for the appearance of fever in malaria

patients. The body temperatures of these patients are irregular over 24 h and can rise as high

as 410C. This periodic appearance of fever is a characteristic of tertian malaria caused by P.

falciparum and P. vivax (Pavithra et al., 2004). The invasion of Plasmodium into new cells is

a highly synchronized event which helps the Plasmodium to evade the immune system of its

host (Garcia et al., 2001).

1.5 Clinical Manifestation of Malaria

The symptoms of malaria appear 10-15 days after infective mosquito bite or as early as seven

days in non-immune individuals. The initial symptoms are generally non specific and mimics

symptoms of systemic viral illness or bacteremia. Severe headache which often heralds the

onset of infection is a characteristic of early symptom in all Plasmodium species that cause

malaria. Other cardinal signs and symptoms include high, spiking fevers (with or without

periodicity), chills, myalgias, malaise and gastro-intestinal symptoms. Placental malaria is

due to P. falciparum adherence to chondroitin sulfate A (CSA) in the placenta. It poses

danger to primigravidae including miscarriage. Malaria is an acute febrile illness and

suspected clinically especially in endemic areas on the basis of fever. P. falciparum malaria

can often progress to severe disease and may lead to organ failure and death if not treated

within 24 h (WHO, 2010; Vinetz et al., 2011; WHO, 2014).

Therefore the goal of treatment of uncomplicated malaria is to cure the infection quickly and

prevent progression to severe illness (WHO, 2010). Severe malaria usually manifests with

one or more of the following symptoms: severe anemia, respiratory distress in relation to

metabolic acidosis, impaired consciousness (cerebral malaria), convulsion (more than two

episodes in 24 h), hypoglycemia and even acute renal failure or acute pulmonary edema

(WHO, 2010). Manifestation of the clinical features depends on some factors which include

previous exposure to malaria infection, degree of immunity acquired by the individual and

other poorly understood parasite factors (Weatherall, 1987).

1.6 Diagnosis of Malaria

The Nigeria National Malaria Treatment Policy Guideline in line with WHO recommends

parasitological confirmation of parasitemia using microscopy or rapid diagnostic tests in all

suspected cases of malaria except in settings where parasitological diagnosis is not possible.

Prompt and accurate diagnosis is paramount in effective management of malaria (WHO,

2010; Federal Ministry of Health, Nigeria, 2011). Considering the fact that malaria is a

potential medical emergency, presumptive diagnosis has been a traditional practice among

clinicians. This is still very challenging due to non specific nature of the signs and symptoms

of malaria which can mimic or mask the symptoms of other potentially life threatening

diseases like viral and bacterial infections or other disease causing febrile illness

(Tangpukdee et al., 2009). Moreover, treating suspected cases of malaria based on clinical

diagnosis alone has led to the overdiagnosis or misdiagnosis of malaria (Reyburn et al., 2004;

Gwer et al., 2007; Nankabirwa et al., 2009; Leslie et al., 2012).

Diagnosis of malaria involves the laboratory investigation and identification of malaria

parasites or antigens in the blood of a patient (Tangpukdee et al., 2009). The diagnostic tools

available for laboratory investigation include the use of the conventional microscopic

confirmation of malaria parasite by staining thick and thin peripheral blood smears (Ngasala

et al., 2008), concentration techniques like rapid diagnostic tests e.g. Paracheck, OptiMAL

(Harvey et al., 2008; Tagbor et al., 2008; Zerpa et al., 2008) and molecular diagnostic

methods like polymerase chain reaction (Holland and Kiechle, 2005; Vo et al., 2007).

The choice of the malaria diagnostic tool can be influenced by malaria endemicity, the

experience of the physician, the urgency of diagnosis, the effectiveness of healthcare

workers and financial resources. In addition to this, the results of the diagnostic tool

especially microscopy is also affected by the competence and expertise of the laboratory

personnel, poor quality of the laboratory reagents, microscope or other equipment,

unpredictable power supply, lack of quality control systems, insufficient supervision and

ambiguous guidelines (Barat et al., 1999; Mboera et al., 2006; Wongsrichanalai et al.,

2007). Whichever method is employed, it must be accompanied by quality assurance.

1.7 Classification of antimalarials

The classification of the antimalarial drugs is based on their activities during their life cycle

as well as by their intended use for chemoprophylaxis or chemotherapy. There are remarkable

differences in the morphology, metabolism and drug sensitivity of the various stages of the

malaria parasite life cycle that occur in humans. There are three main classifications of

antimalarial drugs amidst several generalizations due to the spectrum of activity of these

drugs. The agents used for chemoprophylaxis belong to the first category. The fact that there

is currently no antimalarial drug in practice that can kill sporozoites, shows that it is actually

not possible to prevent malarial infection by administration of drugs to humans.

Chemoprophylactic drugs however, can only prevent the development of symptomatic

malaria caused by the action of the asexual erythrocytic forms of the parasite (Vinetz et al.,

2011).

The second classification is based on the treatment of established infection. The

chemotherapeutic burden of malaria eradication lies in the fact that no single antimalarial is

effective against all liver and intra-erythrocytic stages of the life cycle that may co-exist in

the same patient. This therefore implies that complete eradication of the parasite infection

will require more than one drug. In this class, most commonly used antimalarials such as

artemisinin, chloroquine, mefloquine, quinine and quinidine, pyrimethamine, sulfadoxine and

tetracycline direct their activities against the asexual blood stages that is responsible for the

clinical manifestation of the disease (Vinetz et al., 2011). They are not reliably effective

against primary or latent liver stages. They are however used to treat or prevent clinically

symptomatic malaria. The spectrum of some other antimalarials like atovaquone and

proguanil target not only the asexual erythrocytic forms but also the primary liver stages of P.

falciparum and therefore has a longer postexposure chemoprophylaxis. Another category

comprise solely of primaquine which is most commonly used to eradicate the intra-hepatic

hypnozoite of P. vivax and P. ovale that are responsible for relapsing infections. Even though

primaquine is effective against primary and latent liver stages as well as gametocytes, it is

nevertheless not found to be effective in the treatment of symptomatic malaria (Vinetz et al.,

2011).

The utilization of these antimalarials, despite their anti-parasitic activity for either

chemoprophylaxis or treatment depends on their pharmacokinetics and their safety. For

instance, quinine and quinidine are not used for chemoprophylaxis because they possess

significant toxicity and relatively short half-lives. They are generally reserved for the

treatment of established infections whereas chloroquine which is relatively free from toxicity

and has a long half life is used for chemoprophylaxis in those few areas still reporting

chloroquine sensitive malaria (Vinetz et al., 2011).

1.8 Chemotherapy of Malaria

Therapeutic efficacy is the main determinant of antimalaria treatment policy. Therefore

assessment of antimalaria treatment should be based on parasitological cure rates (WHO,

2010). Any delay in assessing treatment for uncomplicated malaria especially in children less

than 5 years beyond 48 h from the onset of symptoms can progress to severe malaria and

fatality. The actual outcome in severe malaria differs between adult and children but depends

on the nature and degree of organ dysfunction. (Sarkar et al., 2009; von Seidlein, 2012).

Hence early diagnosis and prompt treatment of malaria within 24 h of the onset of symptoms

with an artemisinin-based combination therapy (ACT) is advocated by Roll Back Malaria

Initiative (WHO, 2008c).

Consequently, the primary objective of treating uncomplicated malaria by the Nigerian

National Guidelines for Diagnosis and Treatment of Malaria is to cure the patient rapidly,

prevent further progression to severe disease, and reduce morbidity and mortality. This will

in effect decrease the disease transmission and prevent or delay the emergence of drug

resistance (Nigerian Malaria Treatment Guideline, 2013).

Access to effective therapy is an essential component of a renewed commitment to eradicate

malaria. So far, no malaria vaccine has been successfully developed; therefore chemotherapy

remains the mainstay of malaria control strategies. The recommended treatment of

uncomplicated malaria for children weighing less than 5 kg and pregnant women suffering

from malaria in Nigeria is oral quinine, although Artemisinin-based Combination Therapies

(ACT) may be used after first trimester of pregnancy. The guideline also recommends ACTs

with Artemether/Lumefantrin (AL) as first-line and Artesunate/Amodiaquine (AS/AQ) as

alternative first-line in the treatment of uncomplicated malaria (Nigeria Malaria Treatment

Guideline, 2013). Since malaria is mostly endemic in poor and developing countries, efficacy

of antimalarials is not only put into consideration in the choice of the drugs but also the

affordability, side effects and availability. The drugs used to treat malaria can be grouped as

follows:

4-Aminoquinolones for example; chloroquine, amodiaquine and related compounds

like piperaquine, lumefantrine and halofantrine which are amino alcohol derivatives.

Arylaminoalcohols for example; quinine, mefloquine

8-Aminoquinolones for example; primaquine

Antifolates for example; sulphadoxine/pyrimethamine, dapsone

Antibacterial agents for example tetracyclines, clindamycin.

Artemisinin and its derivatives for example, artemether, artesunate,

dihydroartemisinin, α-β arteether (i.e. the artemisinins).

1.8.1 Chloroquine

Discovery of chloroquine (CQ) in the 1930s revolutionized malaria treatment. It later became

the most widely-used drug from the early 1950s to until the 1990s. Chloroquine which used

to be the drug of choice in the treatment of malaria in many countries afflicted with the

burden is a 4-aminoquinolone with rapid antipyretic and parasiticidal effect (Bell and

Winstanley, 2004).

Chloroquine is a weak base that concentrates in the acidic digestive vacuoles of susceptible

Plasmodium where it binds to ferriproptoporphyrin IX (heme), a by-product of haemoglobin

degradation and disrupts its sequestration. The drug-heme complexes formed, kills the

parasite via oxidative damage to membranes, digestive proteases or other critical

biomolecules. Chloroquine is readily absorbed from the gastrointestinal tract when given

orally and rapidly absorbed as well from intramuscular and subcutaneous sites. It is highly

effective against the erythrocytic forms of P. vivax, P. ovale, P. malariae, P. knowlesi and

chloroquine sensitive strains of P. falciparum (Winstanley et al., 2004; Vinetz et al., 2011).

It was found to be gametocidal and the cost of treatment per adult is less than $0.10 (N20.00)

which made it highly affordable. Pruritus constituted the major adverse effect of CQ and due

to high affinity of CQ to the melanocytes, this effect is mostly pronounced in dark skinned

people. Other adverse effects include hypotension, confusion, convulsion, headache,

gastrointestinal upset among others. Also, retinal toxicity was observed after a long term high

dose therapy (Bell and Winstanley, 2004; Vinetz et al., 2011).

After about ten years of use, mutations within P. falciparum that conferred resistance to CQ

were reported independently in Columbia and Thailand. It was subsequently observed that

CQ-resistant mutations have been spreading quickly through most endemic areas.

Chloroquine clears out resistant parasites less efficiently from the human body than sensitive

(non-resistant) parasites. This resulted in increased morbidity and mortality from malaria

(Kim and Scheider, 2013).

The high rate of chloroquine resistance and associated treatment failure prompted WHO and

many countries in Africa to subsequently withdraw its use and switch to alternative therapies

to treat malaria. Malawi was the first country to replace chloroquine with an antifolate

combination sulphadoxine/pyrimethamine as first-line drug in the treatment of uncomplicated

malaria. Other countries like Kenya, Tanzania, Uganda and even Nigeira among others

followed suit (Kamya et al., 2002; Eriksen et al., 2005; Laufer et al., 2006; Nzila et al.,

2009). The wide distribution, ready availability of chloroquine even in remotest areas and its

affordability may have contributed to its irrational use and rapid spread of resistance.

1.8.2 Amodiaquine

Amodiaquine is also a 4-aminoquinolone and an analog of chloroquine. It results from

incorporation of an aromatic structure into chloroquine’s side chain. It is believed to have

same mechanism of action with chloroquine. Following its biotransformation in the liver after

oral administration, the antimalarial activity is obtained from its conversion into

desethylamodiaquine. This desethyl metabolite of amodiaquine achieves much higher

concentrations than its parent drug. Even though it has cross resistance with CQ it is effective

against chloroquine-resistant strains of P. falciparum. It is reasonably well tolerated. Major

side effects include hepatitis, dizziness (Winstanley et al., 2004; Nosten and White, 2007).

1.8.3 Quinine and quinidine

Quinine and quinidine are the most important of over 20 structurally related cinchona

alkaloids. They differ only in the steric configuration at two of the three asymmetrical

centers. Quinidine is a more potent enantiomer of quinine as an antimalaria and also more

toxic. Quinine, just like chloroquine binds to heme in the digestive vacuole of Plasmodium

and prevents its detoxification.

Quinine is very active against asexual erythrocytic forms of the parasite but due to its short

half life and toxicity it is not used for chemoprophylaxis. It is readily absorbed following oral

or intramuscular administration. It is extensively bound to plasma protein and undergoes

extensive biotransformation in the liver. Quinine and quinidine have been drugs of choice for

the treatment of drug resistant and severe P. falciparum malaria. Their side effects include

cinchonism, hypoglycemia, hypotension, visual and auditory impairment (Winstanley et al.,

2004; Vinetz et al., 2011).

1.8.4 Mefloquine

This is a 4-quinoline methanol with structural similarities to quinine. It also has similar mode

of antimalarial action with chloroquine due to its association with erythrocytic hemozoin. It is

a highly effective blood schizonticide but more effective when combined with an artemisinin

derivative. Due to emergence of multi-drug resistant gene (pfmdr1) mediated by P.

falciparum when used as monotherapy, mefloquine is no longer used as first line treatment of

malaria and also not recommended for recrudescent infections within two months of its

previous use to treat malaria. It is rather recommended when first line treatment fails and for

chemoprophylaxis. It is well tolerated orally. The common side effects are nausea, dizziness,

headache, dysphoria and confusion although acute neuropsychiatric reactions have been

reported and this is more likely to affect young children (Price et al., 1999; Taylor and White,

2004; Vinetz et al., 2011).

1.8.5 Halofantrine

Halofantrine is a phenanthrenemethanol derivative of aminoalcohol. It is related to

mefloquine in activity and is effective against asexual forms of multidrug-resistant P.

falciparum malaria. It undergoes variable and incomplete absorption following oral

administration although its bioavailability increases after ingestion of a fatty meal. It

undergoes biotransformation in the liver to be converted to its active metabolite N-

debutylhalofantrine. The terminal elimination half life is 5 days in individuals with malaria.

Major side effects include gastrointestinal disturbances, orthostatic hypotension and

prolongation of QTc interval. Its cardiotoxic effect is a major concern as it leads to cardiac

arrhythmia which can be fatal (Karbwang and Na-Bangchang, 1994; Winstanley et al., 2004).

In view of the fatal cardiotoxicity caused by halofantrine administration, it is important to

screen patients especially females for contraindications such as underlying heart diseases,

bradycardia, family history of long QT interval, concomitant use of another QT -prolonging

drugs (e.g. mefloquine). It is also critical that halofantrine should be taken only at the

recommended dosage (25 mg/kg) and on empty stomach to minimize the side effects. In fact,

use of halofantrine should be discouraged where safer and efficient antimalarial agents are

available (Bouchaud et al., 2009).

1.8.6 Lumefantrine

Lumefantrine as an aryl amino-alcohol belongs to the same group as mefloquine and

halofantrine and exhibits cross resistance with them. It acts on cardiac myocytes like

halofantrine to block the potassium channels which initiate repolarisation and determine QT

interval but in contrast to halaofantrine, it does not cause prolongation of QTc interval and

no cardiac effects or deaths have been reported. The common side effects are mild and

transient which include headache, dizziness and anorexia. It should be given only in

combination with artemether as Coartem® (Winstanley, 2004; Nosten and White, 2007;

Bouchaud et al., 2009).

1.8.7 Primaquine

Primaquine is an 8-aminoquinolone antimalarial. Even though its mechanism of action has

not been elucidated, in contrast to other antmalarials, primaquine acts on the exoerythrocytic

stage of the parasites in the hepatocytes and therefore it is used to prevent and cure relapsing

malaria caused by P. vivax and P. ovale. Primaquine should be administered simultaneously

with a schizonticidal drug for effective eradication of the erythrocytic stages of Plasmodium.

It is very readily absorbed from the gastrointestinal tract after oral administration. Its adverse

effects include mild anemia, hypertension and anemia (Vinetz et al., 2011).

1.8.8 Sulphadoxine-pyrimethamine

Sulphadoxine-pyrimethamine (SP) is a fixed dose combination of suphonamide and the

antifolate pyrimethamine but not a combination therapy as the two components act on the

same target, folate biosynthesis of the pararsite. Pyrimethamine and the biguanides

(proguanil, chlorproguanil) acts by competitively inhibiting the enzyme dihydrofolate

reductase (DHFR) while suphonamides (sulphadoxine) and sulphones (dapsone) act by

competitively inhibiting the enzyme dihydroptreoate synthetase (DHPS).

Sulphadoxine-pyrimethamine has a long half life. It was used to replace chloroquine and is

still being used especially for prevention of malaria in pregnancy despite the fact that

resistance to SP has also evolved and occurs at high frequency in major malaria endemic

regions. Severe allergic reactions is a well known side effect to sulpha drugs while bone

marrow reactions is associated with pyrimethamine use (Bell and Winstanley, 2004;

Winstanley, 2004; Laxminarayan, 2004).

1.8.9 Antibacterial Agents

Antibiotics including tetracyclines act in the apicoplast (organelles that were discovered in

parasites like Plasmodium which was found to contain its own DNA) by interfering with

protein translation or synthesis. Clindamycin is a lincosamide antibiotics that is effective

against P. falciparum, even as monotherapy. It has mean parasite clearance time of 4 to 6

days and mean fever clearance time of 3 to 5 days. Its combination with quinine reduces its

treatment course from 5 to 3 days with improved adherence. Both tetracycline and

clindamycin are slow-acting. Common side effects of tetracycline and clindamycin include

diarrhea, anorexia, nausea, vomiting and abdominal discomfort. Tetracycline can also cause

teeth discolouration in children less than 8 years old (Winstanley et al., 2004; Obonyo and

Juma, 2012).

1.8.10 Artemisinins

Artemisisnins originate from the plant called sweet wormwood (or sweet Annie: Artemisia

annua). They were first discovered in China more than 1500 years ago as “qinghao” extracts

when it was reported to have antipyretic properties (Woodrow et al., 2005). It was not until

1971 that qinghaosu, a highly active chemical from qinghao was obtained and is now called

artemisinin (Klayman, 1985).

Artemisinin is a sesquiterpene lactone structure purified after extraction from Artemisia

annua plant by crystallization and its antimalarial activity is linked to an endoperoxide

trioxane moiety (Klayman, 1985; Haynes, 2001). Artemisinin is a highly crystalline

compound that is neither soluble in oil or water. It is chemically modified at position C10 to

produce artesunate, artemether, arteether, dihydroartemisinin, and artelinic acid which are all

semi synthetic derivatives of the parent compound. All the derivatives have been variously

formulated for oral, rectal and parenteral administration. They have a broad spectrum of

antimalarial activity and produce the fastest clinical response to treatment (Woodrow et al.,

2005).

1.8.10.1 Antimalarial Properties of Artemisinins

Artemisinins kill all species of plasmodium that infect humans especially P. falciparum and

P. vivax. They have rapid blood schizonticidal characteristics and act on erythrocytic form of

the parasite thereby terminating the clinical manifestations of malaria. They are the fastest

acting antimalarials available. The main antimalarial property of artemisinin has been

attributed to their chemical capability to generate free radicals (such as tert-butylperoxide)

which has been found to kill malaria parasites in comparatively high (mM) concentrations.

Heme-catalyzed cleavage of the peroxidase releases unstable free radicals which malaria

parasites are very sensitive to (Woodrow et al., 2005).

During a blood meal, malaria parasites ingest hemoglobin to release free heme, a complex

moiety of iron-porphyrin which in turn infects the red blood cells. Artemisisnins then react

with Fe2+ by the reductive cleavage of the peroxidase bridge to be converted first into oxygen

centered free radicals and then into carbon centered free radicals by intramolecular hydrogen

abstraction from CH2 groups on the periphery of the artemisinin by the O centered radicals.

This action takes place in the food vacuole of the parasite where either Fe2+ acts as the

catalyst that generates the free radicals from peroxidase structure or ferroprotoporphyrin IX

(reduced haem). Artemisinins also inhibit the malarial parasite’s calcium ATPase

(sarcoplasmic endoplasmic reticulum calcium ATPase, SERCA) as an alternative mechanism

of action (Woodrow et al., 2005). Artemisinin derivatives are all converted into

dihydroartemisinin in-vivo and undergoes metabolic biotransformation with elimination half

lives of about I hour and peak plasma concentration of 4 hours (Nosten and White, 2007).

1.8.10.2 Artemisinin Based Combination Therapy (ACT)

Artemisinin based combination therapy (ACT) is simply the combination of an artemisinin

derivative and another antimalarial which is structurally unrelated but more slowly eliminated

and invariably offer mutual protection to each other. It includes majorly

artemether/lumefantrin, artesunate/mefloquine, artesunate/amodiaquine,

dihydroartemisinin/piperaquine and aretesunate/sulphadoxine-pyrimethamine given over a

three day period. The rationale is based on the same principle or theory underlying the

combination treatments for leprosy, HIV infection, tuberculosis and many cancers where the

individual drug exhibit different modes of action and different mechanisms for resistance

(Nosten and White, 2007).

Monotherapy with artemisinin derivatives have been found to cause approximately 10%

treatment failure even with the advocated 7-day treatment regimen and do not provide post

treatment prophylaxis. A 7-day treatment regimen may lead to poor adherence and

subsequently resistance when there is incomplete treatment (Price et al., 1998; White 1997).

Examples of ACTs include:

Artesunate:

Artesunate can be combined with sulphadoxine/pyrimethamine as an oral dose of 4

mg/kg body weight of artesunate administered once orally for 3 days and a single

oral dose of 25/1.25 mg base/kg body weight of SP. Artesunate is available in a

blister pack of 50 mg while SP is available in a blister pack of 500/25 mg. The same

oral dose of artesunate can be combined with 10 mg/kg oral dose of amodiaquine

given once daily for 3 days but amodiaquine is available in a blister pack of 153 mg

in combination with artesunate and 200 mg as a loose drug (Nosten and White,

2007). Artesunate is also excellently used with mefloquine at a fixed adult dose

combination of 200 mg atesunate and 400 mg mefloquine base or 8.3 mg/kg

mefloquine given orally once daily for 3 days (Taylor and White, 2004).

Artemether

Artemether is combined with lumefantrine as Artemether/lumefantrine (AL) which is

the first fixed dose combination of artemisinin derivative in which the second

unrelated drug is equally active against all human malaria parasites including the

multi-drug resistant P. falciparum. The AL is given as a 6-dose regimen of 80/480

mg at 0, 8, 24, 36, 48 and 60 h for adults weighing 35 kg and above. Fatty meal

enhances its absorption which is also dose limited (Nosten and White, 2007).

Dihydroartemisinin

Dihydroartemisinin/piperaquine is another artemisinine derivative fixed dose

combination drug, commercially available as dihydrartemisinin 40 mg and

piperaquine 320 mg. It is also given orally once daily for 3 days (Nosten and White,

2007).

Artemisinin and its derivatives are generally associated with some gastrointestinal effects like

nausea, vomiting, abdominal pain and diarrhea which are usually mild and self limiting.

There are other artemisinin derivative combination drugs like artesunate/chlorproguanil-

dapsone, artesunate/atovaquone-proguanil as well as artesunate/pyronadrine which are

commonly used outside Africa (Nosten and White, 2007).

1.9 Prevention and Control of Malaria

Resurgence of malaria occurs when control fails. Vectorial capacity plays a major role in

malaria transmission therefore prevention of malaria infections should begin with eliminating

mosquitoes, the vector carrying Plasmodium species that cause malaria and also by a

thorough understanding of the unique behavioral pattern of mosquitoes even before they suck

an infected blood meal. Although mosquitoes have been reported to be nocturnal insects,

blood feeding and resting however occurs in significant proportion outdoors (Reddy et al.,

2011).

Mosquitoes are found everywhere during the day like in schools, markets, offices, hospitals,

inside vehicles among many other places irrespective of continuous in-door spraying. This

adversely affects the indoor-based anti-vector control which is important in the prevention

and control of malaria (Reddy et al., 2011). They also migrate very well at any time from one

community to another and sometimes transported from different vehicles or by perching on

the back of a cyclist when moving from one place to another without being distorted by the

motion of the wind. This migration makes it difficult to control mosquitoes in a community

hence a collaborative approach among communities is advocated. Variation between

countries in epidemiology with appropriate interventions is affected by parasite type, vector

behavior and of course transmission levels (WHO, 2008a).

There seems to be varying characteristics of the Anopheles mosquitoes habiting in Nigeria.

Some of the mosquitoes which operate mostly at night come humming with a buzzing sound.

Some are conspicuously big in size and bite with a strong piercing proboscis while some are

minute with an individual only feeling their presence after him/her has been bitten. Time of

biting also varies among different vector species. In an interesting study by Okwa et al.,

(2009) on transmission dynamics of malaria in four selected ecological zones of Nigeria in

the rainy season, five different species of Anopheles were identified and were found to be

distributed in all the ecological zones of Nigeria. The species include Anopheles gambiae

complex, Anopheles funestus complex, Anopheles moucheti nigeriensis, Anopheles

arabiensis, Anopheles melas. Anopheles gambiae was found to be the most abundant species

while Anophelas melas was the least common species identified.

The use of indoor residual spraying (IRS) and long lasting insecticide treated nets (LLIN) has

actually predisposed certain populations of mosquito to exhibit exophilc behaviours (resting

outdoors) instead of endophlic behaviour therefore affecting the control of mosquitoes. Use

of IRS or LLIN also encourages increased exophagy (feeding outdoors) by mosquitoes in

their biting time (Mathenge et al., 2001; Pates and Curtis 2005; Reddy et al., 2011).

The effectiveness of their use can optimally be achieved if the biting hours of mosquitoes are

put into consideration before use. Their use is nevertheless encouraged because most

mosquitoes migrate indoors late evenings and exit upon appearance of daylight. Nigeria is

richly endowed with diversity in vegetation comprising of mangrove forest and coastal

vegetation, fresh swamp communities, tropical high forest zones, guinea savanna, sudan

savanna and sahel savanna. Each vegetation is also well distributed with different animals

and topographies (Aregheore, 2009). Despite these diversities mosquitoes still thrive well in

all the vegetations all through the seasons, irrespective of their various climatic conditions.

Any approach that will lead to blockage of transmission of malaria parasite to the human host

will definitely be a stepping stone to the complete eradication to malaria. There is currently

no licensed vaccine for the prevention and control of malaria but current candidate vaccine

target just one stage of the parasite’s life cycle. RTS,S/AS01 is a hybrid protein particle that

has reached phase III clinical trials and is undergoing the process of being considered for

licensure following its submission (Hill, 2011).

Chemotherapy with antimalarials significantly contributes to the control of malaria especially

when used for prophylaxis or treatment which consequently prevents morbidity and mortality

from malaria. However, chemoprophylaxis should be considered in individuals at high risks

such as pregnant women and young children. In approaching the targets set by the Roll Back

Malaria Partnership with World Health Organization, many countries in Africa have scaled

up delivery of Intermittent Preventive Therapy (IPT) in pregnant women with a single dose

sulphadoxine plus pyrimethamine (Greenwood, 2010). It is also important to note that

continued use of chemotherapeutic agents to prevent malaria transmission can predispose the

patient to toxicity (Vinetz et al., 2011).

1.10 Review of Plants with Antimalarial Activity

According to WHO consultative group, a medicinal plant can be defined as any plant which

contains in one or more of its organs, substances that can be used for therapeutic purposes or

which are precursors for the synthesis of useful drugs (WHO, 1977). This definition makes it

possible to distinguish medicinal plants whose therapeutic properties and constituents have

been scientifically established and plants that are regarded as medicinal plants but yet to be

validated by a thorough scientific study (Sofowora, 1993).

Since the earliest days of humanity, medicinal plants have formed the basis of healthcare

throughout the world and are still widely used as the recognition for their clinical,

pharmaceutical and economic value is still growing. New medicinal plants are constantly

being discovered and investigated considering the rising cost of prescription drugs and

increased approach to innovation of plant derived drugs. Medicinal plants have been widely

used in virtually all cultures as a source of medicine since times immemorial (Hoareau and

Dasilva, 1999). This is also a common practice in Nigeria where plants are used either as

extracts or infusions in the treatment and management of diseases (Iwu, 1982), they are often

referred to as herbal remedies or ‘concoctions’ and in fact are readily affordable. Limitations

to their use include lack of standardization in storage, dosage, preparations and long term or

short term safety.

The African continent is richly endowed with floral biodiversity with promising

phytochemicals which represents a treasure of useful information in search of antimalarials or

treatments for other diseases. Therefore, the application of ethnopharmacology in the

selection of new plants with antimalarial activity is a logical approach for identification of

new drugs. For thousands of years, plants having constituted the basis for traditional

medicine are good sources of lead compounds for drug development (Onguene et al., 2013).

As far back as the 17th century, quinine a cinchona alkaloid from the bark of cinchona (quina-

quina) tree that belongs to the aryl amino alcohol group of drugs marked the first successful

use of a chemical compound to treat an infectious disease. Quinine remained the mainstay in

malaria treatment until the 1920’s when other more effective synthethic antimalarials were

developed. The most important of the drugs was chloroquine which was extensively used

from the beginning of 1940’s. Quinine was used as a template for the synthesis of

chloroquine and mefloquine (Achan et al., 2011).

More recently the most effective antimalarial in current use, artemisinin is isolated from a

chinese plant Artemisia annua. Various species used traditionally for the treatment of malaria

have been found to belong to certain family of medicinal plants. Among them is

Apocynaceae with reported antimalaria activity of the following, ethanolic stem bark extract

of Alstonia boonei (Iyiola et al., 2011) the seeds, fruits and stem bark of Picralima

nitida (Iwu and Klayman, 1992) and Rauwolfia vomitoria (Agbaje and Elueze, 2006).

Landolphia owariensis being investigated in this study for antimalaria activity of the

methanolic leaf extracts and fractions also belongs to the family of Apocynaceae. So far, over

1200 plant species have been reported in ethnobotanical studies to have antimalarial

activities and are also being investigated for it (Wilcox et al., 2011). Below is a list of plants

with antimalarial activity including the part(s) used (Table 1).

Table 1: List of some plants with antimalarial activity

Name of plant Family Part used

Kaya grandifolia Meliaceae Stem bark

Lowsonia inermis Lythraceae Leaf

Azadirchta indica Meliaceae Stem/leaf

Zingiber officinale Zingiberaceae Root

Striga hermonthica Scrophul oriaceae Whole plant

Tapinanathus sessillifolia Loranthaceae Leaf

Quassia amara Simaroubaceae Leaf

Annona senegalensis Annonacaea Leaf

Cymbogon giganteus Poaceae Leaf

Morinda lucida Rubiaceae Stem bark / leaf

Citrus medica Rutaceae Leaf / flower

Morinda morindiodes Rubiaceae Aerial parts / root bark

Phyllanthus amarus Euphorbiaceae Leaf / stem

Mangifera indica Anacardiaceae Stem bark / leaf

Cajanus cajan Fabaceae Leaf

Vernonia amygdalina Asteraceae Leaf

Acacia nilotica Fabaceae Root

Alstonia Alstonia De Wild Apocynaceae Stem bark / leaf

Source: Ibrahim et al., 2012

1.11 Botanical Profile of Landolphia owariensis

Landolphia belongs to a genus of flowering plants in the family Apocynaceae. The plant is

native to Africa. It was first identified and described in Nigeria as a genus in 1804 by

Landolphia P. Beauv. It subsequently assumed the botanical name Landolphia owariensis P.

Beauv (Okorie, 2014).

1.11.1 Plant Taxonomy

L. owariensis can be identified, classified and described as listed below:

Common names

White rubber vine, vine rubber (English); ‘Ciwoo’ (Hausa); ‘Eso/Utu’ (Igbo); ‘Mba’

(Yoruba) (Burkill, 1985; Owoyele et al., 2001).

Classification

Kingdom: Plantae

Phylum: Magnoliophyta

Class: Magnoliopsida

Order: Gentianales

Family: Apocynaceae

Genus: Landolphia

Species: owariensis (The Global Biodiversity Information Facility, 2013)

Synonyms: Landolphia kirkii, Landolphia petersiana

Kirkii => leaves are glossy green with channeled midrib, flowers are many and white or

creamy-yellow in colour while the fruits are green.

Petersiana => flowers are white and sweetly scented, carried in panicls at the end of the

branches. Fruits are eaten with skin when ripe and without skin when semi-ripe.

Characteristics

Climate: Savannah and tropical rain forest

Habitat: Mesophytes

Habit: liane (Burkill, 1985)

1.11.2 Description of Plant

L. owariensis grows in the savannah as a shrub or a huge liane of secondary deciduous in

dense forests attaining about 100m in height and 1m in girth. The flowers are small and

sweetly scented. The fruits are reddish brown when ripe. They are almost round and about the

size of an orange. The fruit is made of tough skin and a very sweet stringy pulp with

numerous seeds embedded it. It fruits from November to March. L. owaeriensis is the

commonest of the species found in West Africa (Burkill, 1985; Gill, 1992; Owoyele et al.,

2001).

1.11.3 Geographical Distribution of Plant

It is a medicinal plant that occurs in Guinea to West Cameroon, extending across Central

Africa to Sudan, Uganda and Southern Tanganyika. It is associated with tropical forest and

coastal lowland and subtropical bush. It is found either as a sprawling bush or a woody liane

with tendrils (Burkill, 1985; Okonkwo and Osadebe, 2008).

Figure 2: L. owariensis in its natural habitat

1.11.4 Ecology

L. owariensis is found as either a sprawling bush or a woody liane with tendrils depending on

the vegetation. Mahale Mountain region of Western Tanzania is among the geographical

zones where L. owariensis grows. The fruits have been classified as an important food for

chimpanzees at Mahale. The chimpanzees usually swallow the seeds with the fruit pulp then

later defecate the seeds intact in their faeces (Burkill, 1985; Nakamura, 2014).

1.11.5 Ethnomedicinal Uses

Various parts of the plant have been widely used for the treatment of many ailments in Africa

(Okonkwo and Osadebe, 2008). A decoction of the leaves is used as a purgative and to cure

malaria while the root is soaked in local gin for about one week and two full glass of the

resulting alcoholic extract drunk in a day to cure gonorrhea (Gill, 1992). In french Equitorial

Africa, the latex is drunk as enema for intestinal worms while some parts of Ivory Coast use

the latex as an ingredient of arrow poison (Irvine, 1961). It is also used to make native beer

and beverage in Senegal and upper Nile land respectively (Dalziel, 1937).

1.12 Literature Review

Pharmacological screening of the plant indicates that the aqueous, methanol and chloroform

leaf extracts of L. owariensis P. Beauv have moderate analgesic but high anti-inflammatory

activity (Owoyele et al., 2001) as well as antiulcer activities and gastric antisecretory effects

(Olaleye et al., 2008). The leaf extracts of the plant has been found to exhibit antimicrobial

effects (Ebi and Ofoefule, 1997; Okeke et al., 2001; Nwaogu et al., 2007).

It is documented that L. owariensis seeds possessed hepatoprotective activity against

paracetamol-induced (500 mg/kg) hepatopathy in rats (Okonkwo and Osadebe, 2010). This

investigation was done based on the presence of its high antioxidant content (Oke and

Hamburger, 2002). Also, the phytochemical analysis and heavy metals studies of the leaf

extracts indicated the presence of some secondary metabolites and heavy metals (Galadima et

al., 2010).

1.13 Aim

This study is aimed at investigating the antiplasmodial activity of the crude extract and

fractions of the leaves of L. Owariensis P.Beauv in order to validate its traditional use for the

treatment of malaria in South East Nigeria.

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Animals

Adult Swiss albino mice of either gender (25–30g) were used for the study. The mice were

obtained from and housed in the Laboratory Animal Facility of Department of Pharmacology

and Toxicology, University of Nigeria Nsukka. Prior to the study, the animals were allowed

to acclimatize for 7 days in the laboratory. They were fed with normal commercial rodent

pellet diet, given water ad libitum and maintained under laboratory conditions of temperature

(22 ± 1oC), relative humidity (14 ± 1%), and 12 h light and 12 h dark cycle.

All procedures and techniques for the animal experiment were conducted in accordance with

the National Institute of Health Guidelines for the care and use of Laboratory Animals (NIH,

Department of Health Services publication No. 85-23, revised 1985).

2.1.2 Drugs

Chloroquine sulphate BP 400 mg (May & Baker Nigeria PLC).

2.1.3 Chemicals and Reagents

Extraction and fractionation

Analytical grades of methanol, n-hexane, ethyl acetate and silica gel of size 60-120

mesh were obtained from Sigma-Aldrich, Germany.

Pharmacological studies

Tween 80 (Sigma-Aldrich, Germany), distilled water, normal saline, 10% Giemsa

solution, Chloroquine sensitive Plasmodium berghei berghei NK65.

Phytochemical analysis

Ferric chloride, iodide solution, ethanol, dilute ammonia solution, sulphuric acid,

naphthol solution, potassium mercuric iodide solution (Meyer’s reagent), bismuth

potassium iodide solution (Dragendroff’s reagent), Fehling’s solution, Million’s

reagent, picric acid solution.

HPLC analysis

Methanol (HPLC grade), acetonitrile (HPLC grade), Water (HPLC grade) and formic

acid (Analytical grade) were obtained from Sigma-Aldrich, Germany.

2.1.4 Equipment

Rotary vacuum evaporator (Buchi, Switzerland), Whatman filter paper (No 1), measuring

cylinder, spatula, electronic weighing balance (G&G Electronic scale, China), animal

weighing balance, mortar and pestle, beakers, flat bottom conical flasks, glass

chromatographic column, microscope slide, electronic microscope, Shimadzu HPLC system.

2.2 Methods

2.2.1 Collection, Authentication and Preparation of Plant Material

Fresh leaves of Landolphia owariensis were collected from Nsukka, Nigeria in December,

2013. They were identified and authenticated by Mr Ozioko of International Centre for

Ethnomedicine and Drug Development (InterCEDD), Nsukka, Enugu State, Nigeria. A

voucher specimen with number INTERCEDD/067 was deposited at the herbarium of the

centre for future reference.

2.2.2 Preparation of Crude Extract.

The leaves were shade-dried for one week and milled to coarse powder using a mechanical

grinder. The coarse powder obtained (6 kg) was extracted by cold maceration in methanol for

48 h using a mechanical shaker. The resulting mixture was filtered using muslin cloth,

followed by Whatman filter paper (No. 1) and the filtrate concentrated using a rotary

evaporator (40oC). The concentrate was air-dried to a dark brown semi-solid to give the

methanol extract (ME). The yield was 3.67%. The dark brown product was transferred to an

airtight bottle and kept in a refrigerator at 2-8oC till needed. Aliquots of the crude extract

residue was freshly weighed and reconstituted in distilled water before use on each day of the

experiment.

2.2.3 Solvent-guided Fractionation of Crude Extract

The methanol extract (215 g) was mixed with silica gel by triturating in a mortar to obtain a

uniform mix. The mixture was air-dried at room temperature, packed into the

chromatographic glass column and was successively eluted with n-hexane, ethyl acetate and

methanol in the order of increasing solvent hydrophilicity. The fractions were collected and

concentrated in a rotary evaporator (400C) under reduced pressure to obtain n-hexane fraction

(nHF), ethylacetate fraction (EF) and methanol fraction (MF). Aliquots of the fractions were

freshly weighed and reconstituted in 3%v/v Tween 80 before use on each day of the

experiment.

2.2.4 Determination of Yield (%)

The yield of extract and fractions were calculated using the relation:

Yield of extract (%) = weight of extract (g)

weight of plant material macerated (g)X100

Yield of fraction (%) = weight of fraction (g)

weight of extract fractionated (g)X100

2.2.5 Phytochemical Analysis of Extract and Fractions

The extract and fractions were subjected to phytochemical analysis for identification of

constituents using the methods of Harborne (1984).

2.2.5.1 Test for saponins

About 20 ml distilled water was added to 0.25 g of the plant extract (ME) in 100 ml beaker

and boiled gently in a hot water bath for 2 min. The mixture was filtered while hot and

allowed to cool. The filtrate was then diluted with 20 ml of water, shaken vigorously and

observed for the presence of a stable froth upon standing.

2.2.5.2 Test for tannins

Ferric chloride test

About 1.0 g of extract was boiled with 50 ml of water, filtered and used for the ferric chloride

test. Few drops of ferric chloride were added to 3 ml of extract filtrate and the colour of the

resulting precipitate observed. A greenish black precipitate indicates the presence of tannins.

2.2.5.3 Test for flavonoids

Ammonium Test

About 10 ml of ethyl acetate was added to 2 ml of extract and heated on a water bath for 3

min. The mixture was cooled, filtered and filtrate subjected to ammonium test thus: about 4

ml of each filtrate was shaken with 1 ml of dilute ammonium solution. The sugars were

allowed to separate and the yellow colour in the ammoniac layer indicates the presence of

flavonoids.

2.2.5.4 Test for resins

Precipitation test

Alcohol extract obtained by extracting about 0.20 g of each extract with 15 ml of 99%

ethanol was poured into 20 ml of distilled water in abeaker. A precipitate occurring indicates

the presence of resins.

2.2.5.5 Test for steroids and terpenoids

A mixture of about 9 ml of ethanol and 1 ml of the plant extract was concentrated to 2.5 ml

on a boiling waterbath and 5 ml of hot water added. The mixture was allowed to stand for one

hour and the waxy matter filtered off. The filtrate was further extracted with 2.5 ml of

chloroform using separating funnel. To about 0.5 ml of chloroform extract in a test tube was

added carefully 1 ml of concentrated sulphuric acid to form a lower layer. A reddish brown

interface shows the presence of steroids. Another 0.5 ml of the chloroform extract was

evaporated to dryness in a water bath and heated with 3 ml of concentrated sulphuric acid for

10 min. in a water bath. A grey colour indicated the presence of terpenoids.

2.2.5.6 Test for alkaloids

About 20 ml of 5% sulphuric acid in 50% ethanol was added to about 2 g of plant extract and

heated on a boiling water bath for 10 min., cooled and filtered. About 2 ml of the filtrate was

treated with a few drops of Mayer’s reagent, Dragendroff’s reagent, Wagner’s reagent and

picric acid (1%) solution. The remaining filtrate in about 100 ml separating funnel was made

alkaline with dilute ammonia solution, the aqueous alkaline solution was separated and

extracted with two 5 ml portion of dilute sulphuric acid. The extract was tested with a few

drops of Mayer’s, Wagner’s, Dragendroff's reagent and picric acid solution. Alkaloids give

milky precipitate with one drop of Mayer’s reagent, reddish brown precipitate with one drop

of Wagner’s reagent, yellow precipitate with one drop of picric acid solution and brick red

precipitate with one drop of Dragendorf’s reagent.

2.2.5.7 Test for glycosides

Modified Borntrager’s test

About 5 ml of dilute sulphuric acid and ferric chloride solution was added to about 2 ml of

each filtrate, boiled for 5 min, cooled and filtered into a 50 ml separating funnel. The filtrate

was shaken with an equal volume of carbon tetrachloride and the lower organic layer

carefully separated into a test tube. About 5 ml of dilute ammonia solution was added to the

test tube containing each filtrate and then shaken. A rose pink to red colour in the

ammoniacal layer indicates the presence of anthraquinone glycosides.

2.2.5.8 Test for fats and oil

About 0.1 ml of extract was dropped on filter paper and observed. Translucency of the paper

indicates presence of oil.

2.2.5.9 Test for carbohydrates

Iodine test

A drop of iodine solution was mixed with about 0.5 ml of extract. A blue-black colour

indicates the presence of starch.

2.2.5.10 Test for reducing sugars

Fehling’s test

The filtrate obtained after shaking about 1 ml of extract vigorously with about 0.5 ml of

distilled water, was used in the Fehling’s test as follows: to about I ml portion of the filtrate

were added equal volumes of Fehling’s solution A and B boiled on water bath for a few

minutes. A brick red precipitate indicates the presence of reducing sugar.

All the tests for phytochemical analysis were also carried out for the different fractions.

2.2.6 High Performance Liquid Chromatography (HPLC) Analysis

The HPLC analysis was performed with the extract and fractions which exhibited remarkably

high antiplasmodial activity. This comprised of the methanol extract (ME), methanol fraction

(MF) and ethylacetate fraction (EF). Standard procedure was developed for HPLC fingerprint

analysis. The chromatographic system used was Shimadzu HPLC system consisting of Ultra-

Fast LC-20AB prominence equipped with SIL-20AC auto-sampler; DGU-20A3 degasser;

SPD-M20AB UV-diode array detector; column oven CTO-20AC, system controller CBM-

20Alite and Windows LC solution software (Shimadzu Corporation, Kyoto Japan); column,

VP-ODS 5µm, and dimension (150x4.6 mm). The chromatographic conditions included

mobile phase: solvent A: 0.2% v/v formic acid solution; solvent B: acetonitrile; mode:

isocratic; flow rate 0.6 ml/min; injection volume 2 µl of 100 µg/ml solution of extract and 5

µl of 100 µg/ml solution of fraction in mobile phase; detection UV254 nm. The HPLC-

operating conditions were solvent A: 80%; solvent B 20%; Column oven temperature of

40oC. The total run time was 35 minutes.

2.3 Pharmacological Studies

2.3.1 Determination of Acute Toxicity (LD50) of ME

For the acute toxicity test, the animals were fasted overnight, but with access to water ad

libitum and then treated orally with L. owariensis extract. Acute toxicity study was carried

out using the method described by Lorke (1983) with modification. The test involved two

phases.

In the first phase, nine mice were randomly divided into three groups of three mice per group

and given 10, 100 and 1000 mg/kg body weight of ME orally (via a cannula), respectively.

The mice were observed for paw licking, salivation, stretching of the entire body, weakness,

sleep, respiratory distress, coma and death in the first 4 h and subsequently daily for 7 days.

In the second phase, the procedure was repeated using another fresh set of nine mice

randomly divided into three groups of three mice each, and was given 1600, 2900 and 5000

mg extract/kg body weight, respectively. These were observed for signs of toxicity and

mortality for the first critical 4 h and thereafter daily for 7 days. This was recorded and LD50

values was then calculated as the square root of the product of the highest non-lethal dose

(with no deaths) and the lowest lethal dose (where deaths occurred), i.e., the geometric mean

of the consecutive doses for which 0 and 100% survival rates were recorded in the second

phase. The oral median lethal dose was calculated using the formula:

LD50 = √ Maximum tolerated dose x Minimum toxic dose

2.3.2 Rodent Parasite (Plasmodium berghei berghei Nk65)

Chloroquine sensitive Plasmodium berghei berghei NK65 were sourced from National

Institute for Medical Research, Lagos and maintained alive at the Department of

Pharmacology and Toxicology, University of Nigeria, Nsukka, Nigeria by continuous

intraperitoneal passage in mice (Calvalho et al., 1991) after every 5 days. The re-infected

mice were then kept at the Animal Facility Center of Department of Pharmacology and

Toxicology, University of Nigeria Nsukka where the study was carried out. Before the start

of the antiplasmodial study, four of the infected mice were kept and observed to reproduce

signs of diseases such as reduced physical activity, poor feeding, pilor erection and pale

looking tail.

2.3.3 Parasite Inoculation

On Day 1 of each test, the presence of parasitemia was established by microscopic

examination of Giemsa-stained thin blood smear of the donor mouse. Blood was collected

from the tail vein of the donor mouse heavily infected with parasites and diluted with

physiological saline (normal saline) to give a concentration of 108 parasitized erythrocytes

per ml (standard innoculum). Each of the healthy experimental mice was innoculated

intraperitoneally with 0.2 ml of infected blood containing 0.1 x 107 P. berghei berghei

parasitized erythrocytes.

2.3.4 Evaluation of Activity on Early Malaria Infection (4-Day Suppressive

Test)

The Peter’s 4-day suppressive test against chloroquine sensitive Plasmodium berghei

berghei (NK 65) infection in mice was employed (Peters, 1965). A total of eighty-four

healthy experimental mice were inoculated intraperitoneally on the first day (Day 1) as

described above. Each of the infected mice was weighed and randomly divided into fourteen

groups (1-14) of 6 mice per group.

Treatment commenced for all the groups orally, 4 h post inoculation and thereafter 24, 48 and

72 h post inoculation. Group 1, 2 and 3 were treated with graded doses of 200, 400 and 800

mg ME/kg/day orally respectively. The same dose levels were also used to treat groups (4, 5,

and 6), (7, 8 and 9) and (10, 11 and 12) using the fractions nHF, EF and MF for the respective

groups. Group 13 and 14 served as the positive and negative controls, and were given 5

mg/kg/day of chloroquine and 3%v/v Tween 80 respectively. All the groups were treated for

four consecutive days (D1-D4).

On day 5 post innoculation, one drop of blood was taken from the tail of each experimental

mouse and smeared on a microscope slide to make a thin film (Saidu et al., 2000). The thin

films were fixed with methanol, stained with 10% Giemsa solution at pH 7.2 for 10 min and

examined microscopically. Parasitemia level was determined by counting the number of

parasitized erythrocytes out of 100 erythrocytes per field in about 4 random fields under a

light microscope at x100 magnification while average percentage parasitemia suppression

was determined by comparing the parasitemia in the control group with the treated group.

% suppression = Pc − Pt

Pc x 100

Where Pc = average parasitemia in the control group

Pt = average parasitemia in the treated group

2.3.5 Evaluation of Activity on Established Infection (Curative or Rane

Test)

Evaluation of the curative potential of L. owariensis leaf extract was employed using the

method described by Ryley and Peters (1970).

On Day 1 of this test, the presence of parasitemia was established by microscopic

examination of Giemsa-stained thin blood smear of the donor mice heavily infected with

parasites. Thereafter, each of the experimental mice was inoculated as described above.

Seventy two hours after inoculation (D4), the mice were weighed and randomly grouped into

fourteen groups (1-14) of 6 mice per group. Group 1, 2 and 3 were treated with graded doses

of 200, 400 and 800 mg ME/kg/day orally respectively. The same dose levels were also used

to treat groups (4, 5, and 6), (7, 8 and 9) and (10, 11 and 12) using the fractions nHF, EF and

MF for the respective groups. Group 13 and 14 served as the positive and negative controls,

and were given 5 mg/kg/day of chloroquine and 3% Tween 80 respectively. Treatment

continued orally for all the groups for four consecutive days. On each day of treatment, one

drop of blood was collected from the tail of each mouse, smeared unto a microscope slide to

make a thin film, stained with 10% Giemsa stain and examined microscopically at x100

magnification to monitor the parasitaemia level.

Percentage parasitaemia level was determined microscopically on each day by counting the

number of parasitized erythrocytes of approximately 100 erythrocytes per field cells in about

4 random fields under light microscope.

The mean survival time (MST) of the mice in each treatment group was determined by

finding the average survival time (days) of the mice (post innoculum) in each group over a

period of 30 days (D1–D29).

2.3.6 Evaluation of Prophylactic Activity (Repository Test)

The prophylactic activity of the extract was evaluated using the residual infection method

described by Peters (1965). A total of eighty-four healthy mice of both sexes were weighed

and randomly grouped into fourteen groups (1-14) of 6 mice per group. Group 1, 2 and 3

were treated with graded doses of 200, 400 and 800 mg ME/kg/day orally respectively. The

same dose levels were also used to treat groups (4, 5, and 6), (7, 8 and 9) and (10, 11 and 12)

using the fractions nHF, EF and MF for the respective groups. Group 13 and 14 served as the

positive and negative controls, and were given 5 mg/kg/day of chloroquine and 3% Tween 80

respectively. All the groups were treated for four consecutive days (D1-D4). On the fifth day

(D5), all the mice were infected with parasite. At 72 hours (D7) post treatment, blood smears

were made from each mouse (Abatan and Makinde, 1986). Percentage parasitaemia were

then determined microscopically.

2.4 Statistical Analysis

Results were expressed as mean ± standard error of mean (SEM). The results obtained were

analyzed using Statistical Package for Social Sciences (SPSS®) version 21 Inc. Chicago, Ill,

USA. Analysis of Variance (ANOVA) was used to explore the differences among the groups

followed by Dunnet’s post hoc test. Differences between means of treated and control groups

were accepted significant at P˂0.05.

CHAPTER THREE

RESULTS

3.1 Extraction and Fractionation

The extraction yielded 220.19 g of the methanol extract (ME; 3.67% w/w). Fractionation of

215 g of ME yielded 69.466 g of n-hexane (nHF; 32.31% w/w), 45.792 g of ethylacetate (EF;

21.30% w/w), and 41.092 g of methanol (MF; 19.11% w/w) fractions respectively (Table 2).

3.2 Phytochemical analysis of extract and fractions

The ME gave positive results for flavonoids, resins, alkaloids, glycosides, steroids,

terpenoids, fats and oil, tannins, carbohydrate, acidity, protein, reducing sugar and saponins.

The nHF tested positive to resins, steroids, terpenoids and fats and oil. The EF tested positive

to flavonoids, resins, glycosides, steroids, terpenoids, fats and oil, tannins, acidity and

saponins while MF gave positive reactions for flavonoids, alkaloids, glycosides, fats and oils,

tannins, carbohydrate, acidity, protein, reducing sugar and saponins (Table 3).

Table 2: Yield (%) of extract and fractions

Extract Yield (%w/w)

Relative to plant material Relative to ME

ME 3.67 100.00

nHF _ 32.31

EF _ 21.30

MF _ 19.11

ME = methanol extract; nHF = n-hexane fraction; EF = ethylacetate fraction; MF = methanol fraction.

Table 3: Phytochemical constituents of extract and fractions

Phytochemical Relative Presence

constituent ME nHF EF MF

Flavonoids ++ - + + + + + + +

Resins + + + + + + + + -

Alkaloids + + - - + + +

Glycosides + + + - + + + + + +

Steroids + + + + + + + -

Terpenoids + + + + + -

Fats and oil + + + + + +

Tannins + + + + - + + + +

Carbohydrate + + + + - - + + + +

Acidity + + + - + + + +

Protein + + + - - + + +

Reducing

sugar

+ + + - - + + +

Saponins + + - + + + +

ME = methanol extract; nHF = n-hexane fraction; EF = ethylacetate fraction; MF = methanol fraction.

Key

- = Absent

+ = Present in small concentration

+ + = Present in moderately high concentration.

+ + + = Present in very high concentration

+ + + + = Abundantly present.

3.3 High Performance Liquid Chromatography (HPLC) Analysis.

Figures 3, 4 and 5 shows the HPLC profiles of methanol extract (ME), methanol fraction

(MF) and ethylacetate fraction (EF) which indicate their respective component peaks. The

methanol extract of L. owariensis showed nine different components with retention time of

4.05, 4.95, 5.82, 6.93, 7.63, 10.14, 11.91, 13.47 and 18.98 min respectively. The HPLC

fingerprint of ethylacetate fraction showed a total of 10 fingerprints which are 4.02, 5.12,

5.60, 6.00, 7.55, 8.18, 10.82, 12.57, 19.51 and 23.50 min respectively while methanol

fraction revealed a total of 11 fingerprints with the following corresponding peaks 3.61, 3.95,

4.36, 5.06, 5.98, 6.78, 7.26, 8.04, 9.24, 10.68 and 19.35 min.

3.4 Acute toxicity (LD50) of ME

The ME (5000 mg/kg) administered orally did not cause lethality and signs of acute

intoxication after 24 h observation period. The LD50 is therefore greater than 5000 mg/kg

(Table 4).

Figure 3: HPLC fingerprint profile of methanol extract of L. Owariensis

Figure 4: HPLC fingerprint profile of methanol fraction of L. Owariensis

Figure 5: HPLC fingerprint profile of ethylacetate fraction of L. Owariensis

Table 4: Acute toxicity testing of methanol leaf extract of Landolphia owariensis in mice

Phases Dose of extract

(mg/kg)a

Mortality

(Died /used)b

I 10 0/3

100 0/3

1000 0/3

II 1600 0/3

2900 0/3

5000 0/3

a = administered as single phase p.o. b = after 7 days (modification of Lorke (1983). LD50 was

determined to be > 5000 mg/kg.

3.5 Pharmacological studies

3.5.1 Effect of ME and fractions on early malaria infection (4-day

suppressive test)

The methanol crude extract and fractions demonstrated significant P<0.05 and dose-

dependent decrease in parasite count at the experimental doses of 200, 400 and 800 mg/kg

used. The mean percentage chemosuppression produced by ME, nHF, EF and MF were

(39.56, 66.69 and 73.93%), (29.38, 45.81 and 58.75%), (33.75, 46.86 and 58.75%) and

(52.06, 77.06 and 86.44%) respectively while chloroquine at 5 mg/kg body weight produced

a chemosuppression of 77.06% (Table 5).

Table 5: Effects of methanol extract and fractions of Landolphia owariensis on early

infection against Plasmodium berghei berghei infected mice

Treatment

Dose

(mg/kg)p.o.

Parasitemia (%)

Inhibition (%)

ME 200 9.67 ± 0.67* 39.56

ME 400 5.33 ± 0.67* 66.69

ME 800 4.17 ± 0.48* 73.93

nHF 200 11.83 ± 0.79* 29.38

nHF 400 8.67 ± 0.42* 45.81

nHF 800 6.60 ± 0.61* 58.75

EF 200 10.60 ± 0.61* 33.75

EF 400 8.50 ± 0.43* 46.86

EF 800 4.67 ± 0.56* 70.81

MF 200 7.67 ± 0.67* 52.06

MF 400 3.67 ± 0.42* 77.06

MF 800 2.17 ± 0.54* 86.44

CQ 5 3.67 ± 0.42* 77.06

Control - 16.00 ± 0.89 -

n = 6; ME = methanol extract; nHF = n-hexane fraction; EF = ethylacetate fraction; MF = methanol

fraction; CQ = chloroquine; *P<0.05 relative to control (Dunnet’s post hoc).

3.5.2 Effect of ME and fractions of on established infection (Rane test)

The results of the percentage inhibition of parasitemia obtained from this test at the

experimental doses of 200, 400 and 800 mg/kg body weight respectively for ME, nHF, EF

and MF include (69.58, 76.43 and 81.94%), (52.66, 56.27 and 53.04%), (52.93, 60.15,

69.47%) and (73.19, 84.49 and 85.9%) while that of chloroquine at 5 mg/kg was 88.97%

(Tables 6 and 7).

Table 6: Parasitemia measurement of methanol extract and fractions of Landolphia

owariensis on established infection

Dose Parasitemia(%)

Treatment (mg/kg) Day 4 Day 5 Day 6 Day 7 Day 8

ME 200 24.5 ± 1.09 11.8 ± 0.31 8.7 ± 0.42* 6.8 ± 0.43 4.7 ± 0.33*

ME 400 25.8 ± 0.60 10.8 ± 0.31 7.0 ± 0.45* 4.0 ± 0.68 3.0 ± 0.26*

ME 800 20.5 ± 1.06 9.2 ± 0.54* 4.3 ± 0.33* 3.2 ± 0.40* 2.3 ± 0.21*

nHF 200 23.3 ± 1.28 16.8 ± 1.49 13.3 ± 0.56* 11.2 ± 0.60 8.5 ± 0.43*

nHF 400 23.2 ± 1.42 18.7 ± 1.28 12.2 ± 0.60* 9.3 ± 0.42* 5.8 ± 0.47*

nHF 800 26.4 ± 1.08 19.4 ± 0.49 14.4 ± 0.61* 10.6 ± 0.42 5.0 ± 0.37*

EF 200 23.3 ± 1.12 18.2 ± 0.48 13.8 ± 0.60* 10.7 ± 0.49 6.8 ± 0.31*

EF 400 22.3 ± 1.02 15.7 ± 1.38 11.7 ± 1.28* 7.7 ± 0.88 6.8 ± 0.48*

EF 800 21.5 ± 0.99 14.7 ± 0.67 8.7 ± 0.56* 3.7 ± 0.56 5.0 ± 0.37*

MF 200 24.5 ± 1.31 13.2 ± 0.65 7.5 ± 0.61* 4.7 ± 0.67 2.8 ± 0.31*

MF 400 22.2 ± 1.30 8.3 ± 0.72 3.5 ± 0.34* 2.7 ± 0.62 1.8 ± 0.31*

MF 800 23.2 ± 1.14 7.5 ± 1.12 3.7 ± 0.92* 2.3 ± 0.33* 1.3 ± 0.33*

CQ 5 23.5 ± 1.48 6.0 ± 0.63 3.3 ± 0.42* 1.3 ± 0.33 1.0 ± 0.37*

Control - 22.0 ± 1.26 23.8 ± 0.65 26.0 ± 1.59 27.4 ± 1.33 33.60 ± 1.23

n = 6; ME = methanol extract; nHF = n-hexane fraction; EF = ethylacetate fraction; MF = methanol

fraction; CQ = chloroquine; *P<0.05 relative to control (Dunnet’s post hoc).

Table 7: Effect of methanol extract and fractions of Landolphia owariensis on

established infection against Plasmodium berghei berghei infected mice

Treatment Dose

(mg/kg,p.o)

Pre -(D4) Post (D7) treatment % Inhibition Mean

survival time

ME 200 24.5 ± 1.09 4.7 ± 0.33* 80.94 25.67 ± 2.14

ME 400 25.8 ± 0.60 3.0 ± 0.26* 88.37 26.83 ± 2.01

ME 800 20.5 ± 1.06 2.3 ± 0.21* 88.63 26.67 ± 1.69

nHF 200 23.3 ± 1.28 8.5 ± 0.43* 63.57 23.50 ± 2.01

nHF 400 23.2 ± 1.42 5.8 ± 0.47* 74.84 24.67 ± 2.22

nHf 800 26.4 ± 1.08 5.0 ± 0.37* 81.06 25.00 ± 2.00

EF 200 23.3 ± 1.12 6.8 ± 0.31* 70.72 24.00 ± 2.02

EF 400 22.3 ± 1.02 6.8 ± 0.48* 69.41 28.00 ± 0.93

EF 800 21.5 ± 0.99 5.0 ± 0.37* 76.74 28.83 ± 0.83

MF 200 24.5 ± 1.31 2.8 ± 0.31* 88.45 28.50 ± 0.72

MF 400 22.2 ± 1.30 1.8 ± 0.31* 91.75 26.67 ± 2.47

MF 800 23.2 ± 1.14 1.3 ± 0.33* 94.26 27.33 ± 2.67

CQ 5 23.5 ± 1.48 1.0 ± 0.37* 95.74 29.33 ± 0.67

Control - 22.0 ± 1.26 33.6 ± 1.23 - 18.50 ± 2.05

n = 6; ME = methanol extract; nHF = n-hexane fraction; EF = ethylacetate fraction; MF = methanol

fraction; CQ = chloroquine; D4 = day 4; D7 = day 7; *P<0.05 relative to control (Dunnet’s post hoc).

3.5.3 Prophylactic effects of ME and fractions against P. berghei berghei

infected mice

The methanol crude extract and fractions elicited significant (P< 0.05) and dose dependent

decrease in parasite counts. At the test doses of 200, 400 and 800 mg/kg body weight, the

mean percentage suppression of parasitemia produced by ME, nHF , EF and MF were (86.1,

88.69 and 91.58%), (74.7, 82.65 and 85.12%), (76.7, 79.67 and 84.14%) and (89.58, 91.58

and 96.04%) respectively while chloroquine produced mean percentage suppression of

97.02% (Table 8).

Table 8: Effects of methanol extract and fractions on prophylactic infection against

Plasmodium berghei berghei infected mice

Treatment

Dose

(mg/kg)p.o

Parasitemia (%)

% Inhibition

Body weight (g)

Day 1 Day 7

ME 200 4.67 ± 0.33* 86.1 25.85±0.86 28.19±0.64

ME 400 3.8 ± 0.31* 88.69 26.59±1.20 28.74±1.19

ME 800 2.83 ± 0.31* 91.58 24.69±1.56 27.98±1.34

nHF 200 8.5 ± 0.43* 74.7 26.67±2.20 28.38±2.18

nHF 400 5.83 ± 0.48* 82.65 30.51±1.93 32.78±1.99

nHF 800 5.0 ± 0.37* 85.12 30.45±1.90 32.89±1.72

EF 200 7.83 ± 0.31* 76.7 26.10± 1.45 28.73±1.65

EF 400 6.83 ± 0.48* 79.67 29.25±1.58 31.58±1.79

EF 800 5.33 ± 0.33* 84.14 25.29±1.28 28.58±1.30

MF 200 3.5 ± 0.43* 89.58 25.81±1.22 29.27±0.72

MF 400 2.83 ± 0.31* 91.58 28.53±0.98 32.09±1.17

MF 800 1.33 ± 0.33* 96.04 27.44±1.23 31.6±1.02

CQ 5 1.0 ± 0.37* 97.02 28.20±0.79 32.3±0.99

Control - 33.6 ± 1.23* - 30.40±1.82 27.8±1.38

n = 6; ME = methanol extract; nHF = n-hexane fraction; EF = ethylacetate fraction; MF = methanol

fraction; CQ = chloroquine; *P<0.05 relative to control (Dunnet’s post hoc).

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 Discussion

The experiment investigated the antiplasmodial effect of methanol extract and fractions of L.

owariensis leaves in mice. The results obtained from this study showed that the methanol

extract revealed significant antiplasmodial activity in the test models as well as the fractions

which exhibited varying intrinsic antiplasmodial activity in the following increasing order,

nhexane fraction (nHF) < ethylacetate fraction (EF) < methanol extract (ME) < methanol

fraction (MF). The average chemo-suppression produced by the crude methanol extracts and

the fractions were comparable to that of chloroquine used in this study. Several studies too

have utilized chloroquine for predicting treatment outcome in medicinal plants with

suspected antimalarial activity. This is because of the high sensitivity of Plasmodium berghei

to chloroquine (Calvalho, et al., 1991).

Although the signs and symptoms presented by the rodent models is different from that

observed in human plasmodial infection, the rodents present disease features similar to that of

human plasmodial infection (Tomas, et al., 1998). The determination of percentage inhibition

of parasitemia is the most reliable parameter for assessment of antimalaria activity of a

suspected compound. Also, the determination of mean group parasitemia level of less than or

equal to 90% of the mock-treated control animals simply shows that the suspected compound

is active following standard screening studies (Abatan and Makinde, 1986).

The 4 day suppressive test is a standard test commonly used for antimalarial screening.

Methanol extracts and fractions demonstrated significant and dose-dependent decrease in

parasite count at the experimental doses of 200, 400 and 800 mg/kg extract used. This

observation may be due to interchange of phytochemicals which are not all selective against

the malaria parasite. The remarkable chemo-suppression produced by the extract and

fractions on day 4 further supports and favour the ethnomedicinal application of L.

owariensis preparations against malaria in tropical endemic areas (Gill, 1992). The results

obtained from the established infection (curative) shows that the degree of antiplasmodial

activity is directly proportional to the experimental doses used for the methanol extract and

fractions except for n-hexane fraction in which increase in dose did not give a corresponding

increase in antiplasmodial activity though 400 mg/kg and 800 mg/kg revealed better activity

than 200 mg/kg. The prolongation of survival time over 12 days shows the test agents are

active (Peter and Antoli, 1998). This reduced the overall pathologic effects of the parasite on

the mice as seen with the general increase in weight in all the test agents except the untreated

group. The highest chemo-suppression was however seen from the repository tests of both the

methanol extracts and fractions. This observation shows that the extract and fractions were

active against the malaria parasite used for the study and also capable of offering prophylaxis

against new infection although the duration for this effect was not in the scope of this study.

Anaemia, body weight loss and temperature reduction are hallmarks of malaria infection in

animal models, suggesting that an effective plant-derived anti-malarial agent should prevent

body weight loss in Plasmodium infected animals (Langhorne et al., 2002). This was

observed in all the test groups apart from the negative control group in which the test agent

did not cause reduction in weight loss. The observation therefore shows that the extract and

fractions have potentials for effective malaria therapy.

Inhibitory effects on generation of free radicals and haemolysis of red blood cells might be

responsible for the remarkable antiplasmodial activity exhibited by the methanol extract and

fractions (Calvalho, et al., 1991). In addition to this, the presence of secondary metabolites

has been implicated in the antiplasmodial effects of some herbal medicines (Philipson and

Wright, 1990). A lot of studies have shown that presence of alkaloids and terpenoids in

medicinal plants, confer significant promising antimalarial activity in the plants ((Philipson

and Wright, 1990; Onguene, et al., 2013).

The high concentrations of flavonoids, alkaloids, saponins and tannins with abundant

presence of glycosides notably in the methanolic fractions might have contributed to its

highest antiplasmodial activity shown compared to the other fractions and methanol extract.

n-Hexane fraction showed the least activity probably due to the absence of glycosides,

flavonoids, alkaloids and saponins. The chemosuppressive activity in this study was higher

with the more polar test agents (ME and MF) as evidenced by the high concentrations of

phytochemicals. The HPLC fingerprint result revealed that the methanolic fraction contained

more components than the ethylacetate fraction and extract. This suggests that the plant

derives its high chemosuppressive activity from the abundance of components or

phytochemicals in the methanol fraction. All the chromatographic peaks obtained are

reproducible because HPLC fingerprint analysis is specific and can be accurately used for the

authentication and identification of similar or different concentrations of L. owariensis (Giri

et al., 2010). It is also possible that there was protein binding effect since only unbound drug

elicits pharmacological action. Moreover, ethanolic extract of the leaves of L. owariensis was

reported to possess antimicrobial activity (Nwaogu et al., 2007). Agents with such activities

such as tetracycline and its derivatives are employed in the treatment of malaria (Vinetz et

al., 2011). Also, the fact that aqueous, methanol and chloroform leaf extracts of L. owariensis

P. Beauv exhibited moderate analgesic but high anti-inflammatory activity shows that the

plant is capable of suppressing malaria symptoms in patients (Owoyele, et al., 2001). Agents

that possess such activity as well, have been found to provide relief to malaria patients.

(Tijani, et al., 2010). This also supports the fact that pharmacological activity is found in the

more polar components of the plant.

All the tests were performed in vivo because it takes into account possible pro-drug effect

and possible immune system involvement in eradication of infection (Waako, 2005).

Historically, rodent or non-human primate experimental models have been used as long-

standing tools for malaria immunology and pathogenesis research, basic discovery, drug

testing and vaccine development (Langhorne, et al., 2011). There are growing concerns about

the relevance of experimental rodent malaria models in the research community (White, et

al., 2010; Carvalho, 2010; Hunt, et al., 2010). This may be attributed to heterogeneity of

malaria presentation in the various mouse-parasite combinations. Despite these concerns, the

lack of access to human samples and the inability to manipulate the immune response for

mechanistic studies make animal models most appropriate (Langhorne, et al., 2011).

No physical sign of intoxication or mortality was observed in all the experimental animals.

However, the mice that received doses of the extract from 1000 – 5000 mg/kg body weight

portrayed initial signs of restlessness but after one hour, mice in all the groups appeared calm,

drowsy with reduced physical activity and eating. No mortality was recorded during the 24 h

observation period and after 7 days. Absence of death at 5000 mg extract/kg body weight

showed that the LD50 of the methanolic leaf extract is probably greater than 5000 mg/kg body

weight. Based on Lorke’s recommendation (Lorke, 1983), the extract is assumed to be safe

and this was indeed used to determine the experimental doses used in the study.

4.2 CONCLUSION

This study shows that the methanol leaf extract and fractions of L. owariensis posses

antiplasmodial activity against P. berghei berghei infected mice. The active ingredients

responsible for the antiplasmodial activity appear to be contained in the more polar solvent

fraction. The study shows that there is great potential for the leaves of L. owariensis to be

explored for the development of antiplasmodial phytomedicine.

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