ANTI-MALARIA EFFECT OF ETHANOL EXTRACT OF MORINGA …

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ANTI-MALARIA EFFECT OF ETHANOL EXTRACT OF MORINGA

OLEIFERA (AGBAJI) LEAVES ON MALARIA-INDUCED MICE

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR AWARD OF DEGREE OF MASTER OF SCIENCE (M.Sc)

IN PHARMACOLOGICAL BIOCHEMISTRY, UNIVERSITY OF NIGERIA,

NSUKKA

BY

UGWU, OKECHUKWU PAUL-CHIMA

(PG/M.Sc/09/51438)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISOR: PROF. O. F. C. NWODO

SEPTEMBER, 2011

97

CERTIFICATION

Ugwu, Okechukwu Paul-Chima, a postgraduate student of the Department of

Biochemistry with the Reg. No: PG/M.Sc/09/51438 has satisfactorily completed his

requirement for research work for the degree of Master of Science (M.Sc) in

Pharmacological Biochemistry. The work embodied in this project (dissertation) is

original and has not been submitted in part or full for any other diploma or degree of

this or any other University.

PROF. O. F. C. NWODO PROF. L.U.S. EZEANYIKA

(Supervisor) (Head)

EXTERNAL EXAMINER

98

DEDICATION

This work is dedicated to my lovely parents Sir Hyacinth Chima and Lady (Hon.)

Francisca Chika Ugwu.

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ACKNOWLEDGEMENT

Firstly, my sincere gratitude goes to the Almighty God for His love, guidance,

protection, provisions and other countless blessings. My immeasurable gratitude goes

to my supervisor, Prof. O.F.C. Nwodo, for his fatherly support and guidance especially

his painstaking patience during the course of this study in helping to separate the

wheat from the chaff. He inculcated knowledge, wisdom, discipline and humility in me.

I remain grateful Sir. The good Lord will continue to bless and protect you, my Prof. I

appreciate my parents, Sir and Lady Hyacinth Ugwu, for their prayers and support. I

cannot forget my siblings, Adaoma Ugwu, Chinyere Ugwu, Nnenna Ugwu and

Ndidiamaka Ugwu for their encouragements. Also, my unreserved gratitude goes to my

love and wife, Nnenna Jovita Ugwu for her love, prayers and encouragements during

the course of the research. I cannot forget the encouragements of my cousin

Chukwuma Asogwa and my dear friends Sunday Valentine Eze, Emeka Aroh,

Okechukwu Ugwueze , in the course of this research

I also appreciate the encouragements of my distinguished lecturers of

Department of Biochemistry, especially Prof. L.U.S. Ezeanyika, Prof. O. Obidoa, Prof.

I.N.E. Onwurah, Prof. O.U. Njoku, Prof. E.O. Alumanah, Prof. F.C. Chilaka, Prof. P.N.

Uzoegwu, Dr. J.E Parker , Dr. S.O.O. Eze, Dr. B.C. Nwanguma, Dr. V.N. Ogugwa, and all

the tutorial and non tutorial staff of the Department of Biochemistry.

My warm regards equally go to Mr.Thomas Adonu of Adonai Laboratory, Nsukka

and Mr Ikechukwu Eze of Department of Veterinary Medicine, University of Nigeria,

Nsukka.

100

Special thanks also go to my friends Heros, Victor, Paul, Ebube, Dopa, Chris

and other postgraduate colleagues of Department of Biochemistry. Finally, my

immeasurable gratitude goes to my friend Dr. B.O Mama of Department of Civil

Engineering, University of Nigeria, Nsukka, for his financial and moral support during

the course of this research. Thanks to you all and remain blessed.

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ABSTRACT

Percentage parasitaemia, haematological parameters, liver markers, kidney markers, lipid profile of

triacylglycerides, total cholesterol, high density lipoprotein and low density lipoprotein were evaluated

in mice consisting of six groups. Groups 1 (positive control) and 6 (negative control) were treated with

5mg/kg body weight of distilled water, group 5 (standard control) was treated with 5mg/kg body

weight of artesunate while groups 2, 3 and 4 were treated with 45, 90 and 180 mg/kg body weight of

Moringa oleifera ethanol leaf extract. The results showed that percentage parasitaemia of the mice

treated with ethanol leaf extract of moringa oleifera significantly cleared parasitaemia on day 28 of

post treatment in groups 4 and 5 compared with groups 1 (positive control) ,2 and 3. The

haematological parameters of packed cell volume (PCV), haemoglobin concentration of the cell (Hb)

and total red blood cell counts (TRBC) increased significantly (p<0.05) in groups 4 (180 mg/kg body

weight of the extract), group 5 (5 mg/kg body weight of Artesunate) and group 6 (negative control)

compared to group 1 (positive control) on day 28 of post treatment while the haematological parameter

of total white blood cell (TWBC) increased significantly (p<0.05) in groups 3 (90 mg/kg body weight

of the extract) and group 6 (negative control) compared to group 1 (positive control). Kidney marker of

serum creatinine increased significantly (p<0.05) in group 1 (positive control) compared to group 6

(negative control) and other groups. Group 6 (negative control) showed a non-significant difference

(p>0.05) in serum urea compared to group 1 (positive control) and other groups. Liver marker of total

bilirubin (TB) increased significantly (p<0.05) in group 1 (positive control) and group 2 (45 mg/kg

body weight of the extract) compared to group 6 (negative control) and other groups. Alanine

aminotransferase (ALT) also, significantly increased (p<0.05) in group 1 (positive control) and group 2

(45 mg/kg body weight of the extract) when compared to group 6 (negative control). Group 6 (negative

control) showed no significant difference (p<0.05) in aspartate aminotransferase (AST) compared to

group 1 (positive control) and other groups. Alkaline phosphatase (ALP) activity in the mice

significantly increased (p<0.05) in group 1 (positive control) and group 4(180mg/kg body weight of the

extract) compared to group 6 (negative control). Lipid profile of total cholesterol, triacylglycerol, high

density lipoprotein and low density lipoprotein showed non-significant difference (p>0.95) when group

6 (negative control) was compared to group 1 (positive control) and other groups.

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TABLE OF CONTENTS

PAGE

103

Title page … … … … … … … … … … i

Certification … … … … … … … … … … ii

Dedication … … … …. … … … … … … iii

Acknowledgement … … … … … … … … … iv

Abstract … … … … … … … … … vi

Table of contents … … … … … … … … … vii

List of tables … … … … … … … … … x

List of figures … … … … … … … … … xi

List of abbreviations … … … … … … … … … xii

CHAPTER ONE: INTRODUCTION

1.1 Overview of malaria … … … … … … … 2

1.1.1 Signs and symptoms of malaria … … … … … 3

1.1.2 Causes of malaria … … … … … … … … 4

1.1.3 Lifecycle of malaria parasites … … … … … … 5

1.1.4 Recurrent malaria … … … … … … … … 9

1.1.5 Pathogenesis of malaria … … … … … … … 9

1.1.6 Malaria epidemiology … … … … … … … 11

1.1.7 Immunity against malaria … … … … … … … 15

1.1.8 Human genetics and innate resistance … … … … … 16

1.1.9 Diagnosis of malaria … … … … … … … … 17

1.1.9.1 Blood films … … … … … … … … … 17

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1.1.9.2 Molecular methods … … … … … … …

… 18

1.1.10 Prevention and control of malaria … … … … … … 18

1.1.11 Anti-malarial drugs … … … … … … … … 19

1.1.11.1 Chemoprophylaxis … … … … … … … … 22

1.1.12 Drug resistance … … … … … … … … 22

1.1.12.1 Spread of resistance … … … … … … … 24

1.1.12.2 Prevention of resistance … … … … … … … 25

1.2 Moringa oleifera .. … … … … … … … 26

1.2.1 Distribution of Moringa oleifera … … … … … … 28

1.2.2 General nutrition of Moringa oleifera … … … … … 28

1.3 Aim and objectives of the research … … … … … … 29

CHAPTER TWO: MATERIALS AND METHODS

2.1 MATERIALS … … … … … … … … … 30

2.1.1 Animals … … … … … … … … … 30

2.1.2 Moringa oleifera (Agbaji) … … … … … … … 30

2.1.3 Instruments/Equipment … … … … … … … 30

2.1.4 Chemicals/Reagents/Samples … … … … … … 31

2.2 METHODS … … … … … … … … … 31

2.2.1 Extraction … … … … … … … … … 31

2.2.2 Experimental design … … … … … … … 31

2.2.3 Procurement of parasitaemia … … … … … … … 32

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2.2.4 Preparation of EDTA (Sequestrene) anticoagulant … … … … 33

2.2.5 Preparation of giemsa stain … … … … … … … 33

2.2.6 Preparation of alcohol fixative solution … … … … … 33

2.2.7 Methods of Estimations … … … … … … … 33

2.2.7.1 Determination of parasitaemia … … … … … … 33

2.2.7.2 Determination of total red blood cell (RBC) count … … … … 34

2.2.7.3 Determination of total white blood cell (WBC) count … … … 35

2.2.7.4 Determination of packed cell volume (PCV) … … … … … 36

2.2.7.5 Determination of haemoglobin (Hb) concentration … … … … 37

2.2.7.6 Determination of total bilirubin concentration … … … … 38

2.2.7.7 Determination of serum urea concentration … … … … … 39

2.2.7.8 Determination of creatinine concentration … … … … … 40

2.2.7.9 Assay of aspartate aminotransferase (AST) activity … … … … 40

2.2.7.10 Assay of alanine aminotransferase (ALT) activity … … … … 42

2.2.7.11 Assay of alkaline phosphatase (ALP) activity … … … … 43

2.2.7.12 Total cholesterol concentration … … … … … … 44

2.2.7.13 High density lipoproteins (HDL)–cholesterol concentration … … 45

2.2.7.14 Determination of triacylglycerol (TAG) concentration … … … 46

2.2.7.15 Low density lipoprotein (LDL)-cholesterol concentration … … … 47

2.2.7.16 Acute toxicity studies (LD50) … … … … … … 48

2.2.8 Phytochemical Analyses … … … … … … 49

2.2.8.1 Test for carbohydrates … … … … … … … 49

2.2.8.2 Test for alkaloids … … … … … … … 49

2.2.8.3 Test for glycosides … … … … … … … 50

2.2.8.4 Test for saponins … … … … … … … 50

2.2.8.5 Test for tannins … … … … … … … 50

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2.2.8.6 Test for flavonoids … … … … … … … 50

2.2.8.7 Test for resins (Precipitaion Test) … … … … … 51

2.2.8.8 Test for proteins (Million’s Test) … … … …. … 51

2.2.8.9 Test for oils … … … … … … … … 51

2.2.8.10 Test for steroids and terpenoids … … … … … 51

2.2.9 Statistical analysis … … … … … … … 51

CHAPTER THREE: RESULTS

3.1 Phytochemical constituents of Moringa oleifera … … … … 52

3.2 Acute toxicity (LD50) … … … … … … … 53

3.3. Effect of ethanol leaf extract of Moringa oleifera on percentage parasitaemia 54

3.4 Effect of ethanol leaf extract of Moringa oleifera on haemoglobin

concentration … .… … … … … … … 56

3.5 Effect of ethanol leaf extract of Moringa oleifera on total white blood

cell count … … … … … … … … … 58

3.6 Effect of ethanol leaf extract of Moringa oleifera on packed cell

volume … … … … … … … … … 60

3.7 Effect of ethanol leaf extract of Moringa oleifera on red blood

cell count … … … … … … … … … 62

3.8 Effect of ethanol leaf extract of Moringa oleifera on serum creatinine

concentration … … … … … … … … … 64

3.9 Effect of ethanol leaf extract of Moringa oleifera on urea

concentration … … … … … … … … … 66

3.10 Effect of ethanol leaf extract of Moringa oleifera on total bilirubin

concentration … … … … … … … … … 68

3.11 Effect of ethanol leaf extract of Moringa oleifera on

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alanine aminotransferase activity … … … … … …

70

3.12 Effects of ethanol leaf extract of Moninga oleifera on aspartate

aminotrasferase activity … … … … … … … 72

3.13 Effect of ethanol leaf extract of Moringa oleifera on alkaline

phosphatase activity … … … … … … … … 74

3.14 Effect of ethanol leaf extract of Moringa oleifera on total

cholesterol concentration … … … … … … … 76

3.15 Effect of ethanol leaf extract of Moringa oleifera on total high density

lipoprotein concentration … … … … .. … … 78

3.16 Effect of ethanol leaf extract of Moringa oleifera on low density

lipoprotein concentration … … … … … … … 80

3.17 Effect of ethanol leaf extract of Moringa oleifera on triacylglycerol

concentration … … … … … … … … … 82

CHAPTER FOUR: DISCUSSION

4.1 Discussion … … … … … … … … … 84

4.2 Conclusion … … … … … … … … … 90

4.3 Suggestions for further studies … … … … … … 90

References … … … … … … … … … 91

Appendices … … … … … … … … … … 96

108

LIST OF TABLES

109

Table 1: Factors influencing vectorial capacity … … … … …

14

Table 2: Selected anti-malarial drugs … … … … … … 20

Table 3: Scientific classification of Moringa oleifera … … … … 27

Table 4: Phytochemical constituents of Moringa oleifera … … … … 52

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LIST OF FIGURES

Fig. 1 Pictorial view of Moringa oleifera … … … … … … 27

Fig. 2 Effect of ethanol leaf extract of Moringa oleifera on percentage

parasitaemia … … … … … … … … … 55

Fig. 3 Effect of ethanol leaf extract of Moringa oleifera on haemoglobin

concentration … … … … … … … … … 57

Fig. 4 Effect of ethanol leaf extract of Moringa oleifera on total white blood

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cell count … … … … … … … … … 59

Fig. 5 Effect of ethanol leaf extract of Moringa oleifera on packed cell

volume … … … … … … … … … 61

Fig. 6 Effect of ethanol leaf extract of Moringa oleifera on red blood

cell count … … … … … … … … … 63

Fig. 7 Effect of ethanol leaf extract of Moringa oleifera on serum creatinine

concentration … … … … … … … … … 65

Fig. 8 Effect of ethanol leaf extract of Moringa oleifera on urea

concentration … … … … … … … … … 67

Fig. 9 Effect of ethanol leaf extract of Moringa oleifera on total bilirubin

concentration … … … … … … … … … 69

Fig. 10 Effect of ethanol leaf extract of Moringa oleifera on

alanine aminotransferase activity … … … … … … 71

Fig. 11 Effects of ethanol leaf extract of Moringa oleifera on aspartate

aminotransferase activity … … … … … … … 73

Fig. 12 Effect of ethanol leaf extract of Moringa oleifera on alkaline

phosphatase activity … … … … … … … … 75

Fig. 13 Effect of ethanol leaf extract of Moringa oleifera on total

cholesterol concentration … … … … … … … 77

Fig. 14 Effect of ethanol leaf extract of Moringa oleifera on total high density

lipoprotein concentration … … … … .. … … 79

Fig. 15 Effect of ethanol leaf extract of Moringa oleifera on low density

lipoprotein concentration … … … … … … … 81

Fig. 16 Effect of ethanol leaf extract of Moringa oleifera on triacylglycerol

concentration … … … … … … … … … 83

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LIST OF ABBREVIATIONS

ALT Alanine aminotransferase

ALP Alkaline phosphate

AST Aspartate aminotransferase

TB Total bilirubin

Mp Malaria parasite

Hb Heamogloblin

TWBC Total white blood cell count

TRBC Total red blood cell count

TAG Triacylglycerides

CHOL Cholesterol

HDL High-density lipoprotein

LDL Low-density lipoprotein

PCV Packed cell volume

IU/l International unit Per litre

Mmol/l Milimole per litre

µmol/l Micromole per litre

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g/dl Gramme per decilitre

i.p Intraperitoneal

HIV Human immuno deficiency virus

EDTA Ethylene diamine tetra-acetate

SPSS Statistical package for social sciences

CHAPTER ONE

INTRODUCTION

Malaria has been and is still the cause of major human morbidity and mortality (Clark and Cowden,

2003). It is the most important parasitic disease worldwide with an incidence of almost three hundred

million clinical cases and over one million deaths yearly (WHO, 2000). Malaria is directly responsible

for one in five childhood deaths in Africa and indirectly contributes to illnesses and deaths from other

diseases (WHO, 1999). Pregnant women and children under five years of age are the most vulnerable.

In the absence of an effective vaccine, the fight against malaria depends on chemotherapy, the

reduction and prevention of anopheles mosquito contacts with human (Winstainley, 2000). The loss in

effectiveness of chemotherapy due to the emergence of resistant strains, constitutes the greatest threat

to the control of malaria. Therefore, to overcome malaria, new knowledge, products, and tools

especially new drugs are urgently needed (Omulokoli et al., 1997). Traditional methods of treatment

and control of malaria could be a promising source of potential anti-malaria drugs. (Wright and

Phillipson,1990) Moringa oleifera was massively grown and promoted by the local media in Uganda in

the 1980s as a plant which is capable of curing a number of diseases ,including malaria, and of

relieving some symptoms of HIV/AIDS. Moringa oleifera is referred to as a MIRACLE TREE (Fuglie,

2001). This is due to its socio-economic, nutritional, pharmacological and industrial benefits (Makkar

and Becker, 2007). As a result of the impact of malaria on the human race and claimed effectiveness of

Moringa oleifera in curing diseases such as diabetes,typhoid and high blood pressure, it was considered

necessary to investigate the anti-malarial effect of Moringa oleifera.

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1.1 OVERVIEW OF MALARIA

Malaria is a mosquito-borne disease of humans caused by eukaryotic protists of the genus Plasmodium.

It is transmitted from one human to another by a bite of an infected female anopheles mosquito. It is

widespread in tropical and sub-tropical regions, including much of Sub-Sahara Africa, Asia and the

Americas (Clark and Cowden, 2003). Plasmodium species are generally host specific and vector

specific in that each species will only infect a limited range of hosts and vectors. Four species of

plasmodium can infect and be transmitted by humans. They are Plasmodium falciparum, Plasmodium

vivax, Plasmodium ovale and Plasmodium malariae. Malaria caused by Plasmodium vivax,

Plasmodium ovale, and Plasmodium malariae is generally milder and rarely fatal. The fifth species,

Plasmodium knowlesi is a zoonosis that causes malaria in Macaques but can also infect humans.Severe

disease results largely from Plasmodium falciparum.

In humans, the parasites called sporozoites travel to the liver, where they mature and release another

form, the merozoites. These enter the bloodstream and infect the red blood cells. The parasites multiply

inside the red blood cells, which then ruptures after 48 to 78 hours, infecting more red blood cells

(Trampuz et al., 2003). The first symptoms usually occur 10 days to 4 weeks after infection, though

they can appear as early as 8 days or as long as a year after infection. The symptoms occur in cycles of

48 to 72 hours.

The majority of symptoms are caused by the massive release of merozoites into the bloodstream, the

anaemia resulting from the destruction of the red blood cells and the problems caused by large amount

of free hemoglobin released into circulation after red blood cells rupture. Malaria can also be

transmitted from a mother to her unborn baby (congenitally) and through blood transmission (Clark and

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Cowden, 2003). Malaria is transmitted by mosquitoes in temperate climates, but the parasites

disappear over the winter. The disease is a major health problem in most of the tropics and sub-tropics

(Clark and Cowden, 2003). WHO (2005) estimates that there are three hundred to five hundred million

cases of malaria each year and more than one million people die. It presents a major health hazard for

travelers to warm climates. In some areas of the world, mosquitoes that transmit malaria have

developed resistance to insecticides. In addition, the parasites have developed resistance to some

antibiotics. This has led to difficulties in controlling both the rate of infection and the spread of the

disease.

1.1.1 Signs and Symptoms of Malaria

Symptoms of malaria include flu-like illness with fever, chills, muscle aches and headache. Some

patients develop nausea, vomiting, cough and diarrhoea. Cycles of chills, fever and sweating that repeat

every one, two or three days are typical. There can be sometimes vomiting, diarrhoea, coughing and

yellowing (jaundice) of the skin and whitening of the eyes due to destruction of red blood and liver

cells (Mueller et al., 2007). People with severe Plasmodium falciparum malaria can develop bleeding

problems, shocks, liver and kidney failure, central nervous system problems and they can die from

infection or its complications. Celebral malaria (coma, altered mental status or seizures) can occur with

severe Plasmodium falciparum infection. It can be lethal if not treated quickly. Even with treatment,

about 15 -20% die (Adams et al., 2002). The symptoms can be summarized as follows:

i. Anaemia

ii. Chills

iii. Coma

iv. Convulsion

v. Fever

vi. Headache

vii. Jaundice

viii. Muscle pain and Nausea

ix. Bloody stools

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x. Sweating and vomiting.

The classic symptoms of malaria is cyclical occurrence of sudden coldness followed by rigour, then

fever and sweating lasting four to six hours, occurring every two days in Plasmodium vivax and

Plasmodium ovale infections, while every three days for Plasmodium malariae. Malaria due to

Plasmodium falciparum can give recurrent fever every 36 – 48 hours or a less pronounced and almost

continous fever. For reasons that are poorly understood, but that may be related to high intracranial

pressure, children with malaria frequently exhibit abnormal posturing, a sign indicating severe brain

damage (Idro et al., 2005). Malaria has been also found to cause cognitive impairments, especially in

children. It causes widespread anaemia during a period of rapid brain development and also direct brain

damage. The neurologic damage results from celebral malaria in which children are more vulnerable

(Trampuz et al., 2003). Celebral Malaria is associated with retinal whitening, which may be a useful

clinical sign in distinguishing between malaria and other causes of fever (Trampuz, et al., 2003).

Severe malaria is almost exclusively caused by Plasmodium falciparum infection and usually arises 6 –

14 days after infection.

Consequences of severe malaria include coma and death if untreated. Young children and pregnant

women are more vulnerable. Splenomegaly (enlarged spleen), severe headache, celebral ischemia,

hepatomegally (enlarged liver), hypoglycemia and hemoglobinuria with renal failure may occur. Renal

failure is a feature of blackwater fever, where hemoglobin from lysed red blood cells leak into the

urine. Severe malaria can progress extremely rapidly and cause death within hours or days (Makintosh

et al., 2004). In most severe cases of the disease, fatality rate can exceed 20% even with intensive care

and treatment (Makintosh et al., 2004). In endemic areas, treatment is often less satisfactory and the

overall fatality rate for all cases of malaria can be as high as one in ten (Trampuz et al., 2003).The long

term developmental impairments have been documented in children who have suffered episodes of

severe malaria.

1.1.4 Causes of Malaria

Malaria is caused by a parasite that is transmitted by the bite of an infected female anopheles mosquito.

Malaria is caused by the members of the genus Plasmodium (Phylum Apicomplexan).

Plasmodium falciparum is the most common cause of infection and is responsible for about 80% of all

malaria cases, and it is also responsible for about 90% of the deaths from malaria (Gardener et al.,

1998). Parasitic Plasmodium species can also infect birds, reptiles, monkeys, mice, chimpanzees and

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rodents. There have been documented human infections with severe Simian species of malaria,

namely: Plasmodium knowlesi, Plasmodium inui, Plasmodium cynomolgi, plasmodium simiovale,

Plasmodium brazilianum, Plasmodium schwetzi and Plasmodium simium; However, with the exception

of Plasmodium knowlesi, these are mostly of limited public health importance (Redd et al., 2006).

Malaria parasites have apicoplasts, an organelle usually found in plants. These apicoplasts are thought

to have originated through the endosymbiosis of algae and play a crucial role in various aspects of

parasite metabolism, e.g fatty acid biosynthesis. To date, 466 proteins have been found to be produced

by apicoplasts and these are now being looked at as possible targets for novel anti-malarial drugs

(Pasvol, 2006).

1.1.5 Lifecycle of Malaria Parasites

Human and other mammalian plasmodium species are transmitted by anopheline mosquitoes. The

parasite is injected with the saliva during mosquito feeding and first undergoes a round of merogony in

the erythrocytes. Gametogony begins within the erythrocytes of the vertebrate host and is completed

within the mosquito where sporogony takes place. This lifecycle exhibits the general features of other

apicomplexan parasites characterized by asexual replication and the formation of invasive stages with

typical organelles (Trampuz et al., 2003). The cycle is divided into various stages such as:

i. LIVER STAGE: Human infection is initiated when sporozoites are injected with the saliva during

mosquito feeding. The sporozoites enter the circulatory system and within 30-60 minutes will

invade a liver cell. The host cell entry, as in all apicomplexan is facilitated by apical organelles

(Mueller et al., 2007). After invading the hepatocyte, the parasite undergoes an asexual replication.

This replicative stage is often called exoerythrocytic or pre-erythrocytic schizogony. Schizogony

refers to a replicative process in which the parasite undergoes multiple rounds of nuclear division

without cytoplasmic division followed by budding or segmentation to form a progeny. The

progeny, called merozoites , are released into the circulatory system following rupture of the host

hepatocyte. In Plasmodium vivax and Plasmodium ovale, some of the sporozoites do not

immediately undergo asexual replication, but enter a dormant phase known as the hypnozoite. This

hyponozoite can reactivate and undergo schizogony at a later time resulting in a relapse (Idro et al.,

2005). Relapse has a specific meaning in regards to malaria and refers to the reactivation of the

infection via hypnozoites. Recrudescence is used to describe the situation in which parasitemia falls

below detectable level and later increases to a patent level. Interestingly, strains isolated from

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temperate regions tend to exhibit a longer latent period between the primary infection and the

first relapse than strains from tropical regions with continuous transmission (Idro et al., 2005).

ii. BLOOD STAGE: Merozoites released from the infected liver cells invade erythrocytes. The

merozoites recognize specific proteins on the surface of the erythrocyte and actively invade the cell

in a manner similar to other apicomplexan parasites. After entering the erythrocyte, the parasite

undergoes a trophic period followed by an asexual replication. The young trophozoite is often

called a ring form due to its morphology in giemsa stained blood smears. As the parasite increases

in size, this ring morphology disappears and it is called the trophozoites. During the trophic period

the parasite ingests the host cell cytoplasm and breaks down the hemoglobin into amino-acids. A

by-product of the hemoglobin digestion is the malaria pigment or hemozoin. These golden-brown

to black granules have been long recognized as distinctive feature of blood-stage parasites

(Mackintosh et al., 2004). Nuclear division marks the end of the trophozoite stage and the

beginning of the schizont stage. Erythrocytic schizogony consists of 3 – 5 rounds of nuclear

replication followed by budding process. Late stage schizonts in which the individual merozoite

becomes discernable are called segmenters. The host erythrocytes rupture and release the

merozoites. These merozoites invade new erythrocytes and initiate another round of schizogony.

The blood-stage parasites within the host usually undergo a synchronous schizogony. The

simultaneous rupture of the infected erythrocytes and the concomitant release of antigens and waste

products accounts for the intermittent fever paroxysms associated with malaria (Makintosh et al.,

2004). The blood stage schizogony in Plasmodium falciparum differs from the other human

malarial parasites in that trophozoite and schizont infected erythrocytes adhere to capillary

endothelial cells and are not found in the peripheral circulation. This sequestration is also

associated with celebral malaria (Mueller et al., 2007).

iii. SEXUAL STAGE: As an alternative to schizogony, some parasites undergo a sexual cycle and

terminally differentiate into either micro or macrogametocytes. The factors involved in the

induction of the gametocytogenesis are not known. However, commitment to the sexual stage

occurs during the asexual erythrocytic cycle that immediately preceed gametocyte formations.

Daughter merozoites from this schizont will develop into either all asexual forms or all sexual

forms.

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Gametocytes do not cause pathology in the human host and will disappear from the circulation

if not taken up by a mosquito (Wellems, 2002). Gametogenesis or the formation of micro and

macrogametes, is induced when the gametocytes are ingested by a mosquito. After ingestion by the

mosquito, the microgametocyte undergoes three rounds of nuclear replication (Phillip and Nicky,

2010). The macrogametocytes mature into macrogametes. The high mobile microgametes will seek

out and fuse with a macrogamete. Within 12 – 24hours, the resulting zygote develops into ookinete.

The ookinete is a motile invasive stage which transverses both the peritrophic matrix and the

midgut epithelium of the mosquito. The invasion process is similar to other apicomplexans except

that the ookinete does not have rhoptries and does not form a parasitophorous vacuole after

invading the host cell (Mueller et al., 2007).

iv. SPOROGONY: After reaching the extracellular space between the epithelial cells and the basal

lamina, the ookinete develops into oocyst (Talman et al., 2004). The oocysts undergo an asexual

replication called sporogony, which culminates in the production of several thousand sporozoites.

This generally takes 10 – 28 days depending on species and temperature. Upon maturity, the oocyst

ruptures and releases the sporozoites which cross the basal lamina into the hemocoel (body cavity)

of the mosquito (Talman et al., 2004). These sporozoites are motile and have ability to specifically

recognize the salivary glands. After finding the salivary glands, the sporozoites will invade and

transverse the salivary gland epithelial cells and come to lie within its lumen. Some of these

sporozoites will be expelled into the vertebrate host as the mosquito takes a blood meal, and thus

re-initiate the infection in the vertebrate host. Although, the hemocoel and salivary gland

sporozoites are morphologically similar, they are functionally distinct. Salivary gland sporozoites

efficiently invade liver cells, but cannot re-invade the salivary glands, whereas the hemocoel

sporozoites are inefficient at invading liver cells.

Finally, malaria parasite exhibits a lifecycle with a typical apicomplexan features. There are three

distinct invasive stages: sporozoites, merozoites and ookinete. All of them are characterized by

apical organelles and can invade or pass through host cells (Philip and Nicky, 2010).Two distinct

merogony are observed. The first, called exoerythrocytic schizogony, occurs in the liver and is

initiated by the sporozoites. The resulting merozoites then invade erythrocytes and undergo

repeated rounds of merogony called erythrocytic schizogony. Some of the merozoites produced

from the erythrocytes schizogony will undergo gamogony. Plasmodium gamogony is described in

two phases: Gametocytogenesis occurring in the blood stream of the vertebrate host and

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gametogenesis taking place in the mosquito gut. The gametes fuse to become a zygote which

first develops into an ookinete and then becomes an oocyst where sporogony takes place (Trampuz

et al., 2003). The parasite’s secondary (intermediate) hosts are humans and other vertebrates.

Female mosquitoes of the anopheles genus are the primary hosts and transmission vectors. Young

mosquitoes first ingest the malaria parasite by feeding on an infected human carrier and the infected

anopheles mosquito carries the plasmodium sporozoites in their salivary glands. A mosquito

becomes infected when it takes a blood meal from an infected human (Biovin, 2002). Once

ingested, the parasite gametocytes taken up in the blood will further differentiate into male or

female gametes and fuse in the mosquito gut. This produces an ookinete that penetrates the gut

lining and produces an oocyst in the gut wall. When the oocyst ruptures, it releases sporozoites that

migrate through the mosquito’s body to the salivary glands, where they are then ready to infect a

new human host (Biovin, 2002). This type of transmission is occasionally referred to as anterior

station transfer (Biovin, 2002). The sporozoites are injected into the skin, alongside saliva, when

the mosquito takes a subsequent blood meal. Only the female mosquitoes feed on blood while male

mosquitoes feed on plant nectar (Trampuz et al., 2003). So, male mosquitoes do not transmit

diseases. The female of the anopheles genus of the mosquito prefer to feed at night. They usually

start searching for a meal at dusk and will continue throughout the night until they take a meal.

When an infected female anopheles mosquito bites a person and injects the malaria parasite

(sporozoites) into the blood, the sporozoite passes through the blood stream to the liver where they

mature and eventually infect the human red blood cells. While in the human red bood cells, they

develop until, an uninfected mosquito takes a blood meal from an infected human and ingests the

human red blood cells with the parasite. Then, the parasites enter the anopheles mosquito’s stomach

and eventually invade the mosquito salivary glands and the cycle continues (Trampuz et al., 2003).

1.1.4 Recurrent Malaria

Malaria recurs after treatments for two reasons. Recrudescence occurs when parasites are not cleared

by treatment (Dondorp et al., 2010). Relapse is specific to Plasmodium vivax and Plasmodium ovale

and involves re-emergence of blood stage parasites from latent parasites (hypnozoites) in the liver.

1.2.5 Pathogenesis of Malaria

Malaria develops via two phases: an exoerythrocytic phase and an erythrocytic phase. The

exoerythrocytic phase involves infection of the hepatic system or the liver, whereas the erythrocytic

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phase involves the infection of the erythrocytes or the red blood cells (Trampuz et al., 2003).

When an infected mosquito pierces a person’s skin to take blood meal, sporozoites in the mosquito

saliva enter the blood stream and migrate to the liver. Within minutes of being introduced into the

human host, the sporozoites infect hepatocytes, multiplying asexually and asymptomatically for a

period of 8 – 30 days (Idro et al., 2005). Once in the liver, these organisms differentiate to yield

thousands of merozoites, which favours the rupture of their host cells, thereby escaping into the blood

and infect red blood cells, thus beginning the erythrocytic stage of the lifecycle. The parasite escapes

from the liver undetected by wrapping itself in the cell membrane of the infected host liver cell (Idro et

al., 2005). Within the red blood cells, the parasites multiply further, again asexually, periodically

breaking out of their hosts to invade fresh red blood cells. Thus, classic descriptions of waves of fever

arise from simultaneous waves of merozoites escaping and infecting the red blood cells. Some

Plasmodium vivax and Plasmodium ovale sporozoites do not immediately develop into exoerythrocytic

phase merozoites, but instead produce hypnozoites that remain dormant for periods ranging from

several months (6 – 12 months is typical) to as long as three years. After a period of dormancy, they

reactivate and produce merozoites. Hypnozoites are responsible for long incubation and late relapses in

these two species of malaria (Makintosh et al., 2004). The parasite is relatively protected from attack

by the body’s immune system because for most of its human lifecycle, it resides within the liver and

blood cells and is relatively invisible to immune surveillance.

However, circulating infected blood cells are destroyed in the spleen. To avoid this fate, the

Plasmodium falciparum parasite displays adhesive proteins on the surface of the infected blood cells,

causing the blood cells to stick to the walls of small blood vessels, thereby sequestering the parasite

from passage through the general circulation and spleen (Idro et al., 2005). This stickiness is the main

factor giving rise to hemorrhagic complications of malaria. High endothelial venules (smallest branches

of the circulatory system) can be blocked by the attachment of masses of these infected red blood cells.

The blockage of these vessels causes symptoms such as in placetal and celebral malaria. In celebral

malaria, the sequestrated red blood cells can breach the brain barrier possibly leading to coma

(Williams, 2006).

Pathology associated with all malarial species is related to the rupture of the infected erythrocytes and

the release of parasite material and metabolites, hemozoin (malaria pigment) and cellular debris (Idro et

al., 2005). The deposition of hemozoin has been known as a characteristic feature of malaria. There is

an increased activity of the reticuloendothelial system, particularly in the liver and spleen and thus their

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enlargement, as evidenced by macrophages with ingested infected and normal erythrocytes.Except

for Plasmodium falciparum, the pathology associated with malaria tends to be benign. Several severe

complications can be associated with Falciparum malaria with celebral malaria being the most notable

and a frequent cause of death.

Celebral malaria is characterized by an impaired consciousness. The presenting symptoms are severe

headache followed by drowsiness, confusion and ultimately coma. Convulsions are also frequently

associated with celebral malaria. These neurological manifestations are believed to be due to the

sequestration of the infected erythrocytes in the celebral micro-vasculature. Sequestration refers to the

cytoadherence of trophozoite and schizont erythrocytes to endothelial cells of deep vascular beds in

vital organs, especially brain, lung, gut, heart and placenta. This sequestration provides several

advantages for the parasite. The major advantage is the avoidance of the spleen and the subsequent

elimination of infected erythrocytes. Cytoadherence appears to be mediated by the electron- dense

protuberances on the surface of the infected erythrocyte (Idro et al., 2005). These knobs are expressed

during the schizont and trophozoite stages. Among human plasmodium species, knobs are restricted to

Plasmodium falciparum and their presence might indicate that they play a role in cytoadherance.

Electron microscopy also shows that the knobs are contact points between the infected erythrocytes and

the endothelial cell. As stated earlier, Plasmodium falciparum causes the most severe form of malaria

in humans with one to three million deaths annually (Clark and Cowden, 2003). The multiplication of

the parasite inside red blood cells is responsible for its severity and mortality that are associated with

the disease. After the parasite invasion, the red blood cells undergo profound structural and

morphological changes, thereby altering their physical properties and impairing circulation. In contrast

to normal red blood cells, parasitized cells become rigid and adhere to the lining of the blood vessels

and other cell types (Trampuz et al., 2003). Those changes are known to be caused by proteins the

parasite produce inside the cells of its host and export across several membranes out to the red blood

cell itself. Earlier studies showed two important ingredients: Plasmodium falciparum erythrocyte

membrane protein (PFEMP1), which allows infected cells to stick to blood vessels, and knobs made up

of a second protein knob associated histidine – rich protein (KAHRP) that anchor (PFEMP1) at the red

blood cell surface (Clark and Cowden, 2003).

1.2.6 Malaria Epidemiology

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Malaria is primarily a disease of the tropics and subtropics (Samba, 1997). It is widespread in hot

humid regions of Africa, Asia, South and Central America (Clark and Cowden, 2003). The disease was

also common in many temperate areas including the U.S.A, Europe and Northern Eurasia and Asia, but

has been eradicated. But many areas which previously had malaria under control, are experiencing a

resurgence (Cox, 2002).The four human malarial species exhibit an overlapping geographical

distribution. Plasmodium vivax and Plasmodium falciparum are the most commonly encountered

species with Plasmodium vivax being the most widespread geographically. Mixed infections are found

mainly in endemic areas. Molecular methods suggest that Plasmodium malariae and plasmodium ovale

might be more widespread and prevalent than previously thought (Mueller et al., 2007). The

epidemiology of malaria can be viewed in terms of being stable or endemic and unstable or epidemic.

Stable malaria refers to a situation in which there is a measureable incidence of natural transmission

over several years. This would also include areas which experience seasonal transmission. Different

areas can experience different level of incidence rates and this is often denoted by: hypoendemic,

mesoendemic and hyperendemic. Persons living in highly endemic areas usually exhibit a high level of

immunity thereby being able to tolerate the infection well.

Unstable or epidemic malaria refers to an increase in malaria in areas of low endemic or outbreak in

areas previously without malaria or among non-immune persons. These outbreaks can usually be

attributed to changes in human behaviour or effects on the environment. For example, human migration

and resettlement can either introduce malaria into an area or expose a previously non-immune

population to endemic transmission.

Changes in the ecology caused by natural disasters or public work projects such as construction of road

can also impact malaria transmission and lead to epidemics (Mueller et al., 2007).

It is estimated that malaria causes two hundred and fifty million cases of fever and approximately one

million deaths annually (Kilama and Ntoumi, 2009). The vast majority of cases occur in children under

5 years of age, pregnant women are also vulnerable. Despite efforts to reduce transmission and increase

treatments, there has been little change in the areas that are at risk of this disease since 1992.

Indeed, if the prevalence of malaria stays on its present upward course, the death rate could double in

the next twenty years (Humpherys, 2001). Precise statistics are unknown because many cases occur in

rural areas where people do not have access to hospital or the means to afford healthcare. As a

consequence, the majority of cases are undocumented (Humpherys, 2001).

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Although co-infection with HIV and malaria do cause increased mortality, this is less of a problem

than with HIV/tuberclosis co-infection, due to that, the two diseases usually attack different age-range,

with malaria being most common in the old (Sachs and Malaney, 2002). HIV/malaria co-infection

produces less severe symptoms than the interaction between HIV and tuberculosis, HIV and malaria do

contribute to each other’s spread. This effect comes from malaria increasing the viral load and HIV

infection increasing a person’s susceptibility to malaria infection (Mackintosh et al., 2004).

Malaria is presently endemic in a broad band around the equator, in areas of the Americas, many parts

of Asia, and more of Africa. However, it is in sub-saharan Africa that 85-90% of malaria fatalities

occur (Sachs and Malaney, 2002). The geographical distribution of malaria within large regions is

complex with malaria afflicted and malaria-free areas being often found close to each other

(Humpherys, 2001). In drier areas, outbreaks of malaria can be predicted with reasonable accuracy by

mapping rainfall. Malaria is more common in rural areas than in cities; this is in contrast to dengue

fever where urban areas present the greater risk (James and Webb, 2009). For example, several cities in

Vietnam, Loas and Cambodia are essentially free from malaria, but the disease is present in many rural

regions (James and Webb, 2009). By contrast, in Africa, malaria is present in both rural and urban

areas, though the risk is lower in larger cities (James and Webb, 2009). The global endemic levels of

malaria have not been mapped since the 1960s.

However, the Welcome Trust UK, has funded the malaria Atlas Project to rectify this, thereby

providing a more contemporary and robust means with which to assess current and future malaria

disease burden. The intricate interactions between host, parasite, and vector are the major factors in this

epidemiological complexity. For example, as with all vector transmitted diseases, the parasite must be

able to establish a chronic infection within the host to maximize the opportunities for transmission

(Mueller et al., 2007). This is especially true in the case of low endemicity. In general, malaria

infections are characterized by an initial acute phase followed by a longer relatively asymptomatic

chronic phase. This is due, in part, to the ability of the parasite to avoid complete clearance by the

immune system. Plasmodium falciparum exhibits an antigenic variation that allows it to stay ahead of

immune system. Plasmodium vivax and Plasmodium ovale exhibit the hypnozoite stage and are capable

of relapses. This allows the parasite to maintain the infection within the human host even after the

blood stage of the infection has been cleared. The relative long interval between relapses in some

Plasmodium vivax isolates probably explains its ability to maintain transmission cycles in some

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temperate climates. Some molecular epidemiology studies have indicated that Plasmodium

falciparum can also produce long-term chronic infections.

However, in regards to the host, humans are the major reservoir for the parasite and sustained

transmission depends upon maintaining a pool of infected individuals and contact between humans and

anopheline mosquitoes (Miller et al., 2002). Several factors influence the susceptibility of humans to

infection. Obviously the immune status of the individual and their prior experience with malaria will

influence the course of the infection. Pregnant women, especially during the first pregnancy are

susceptible to Falciparum malaria as illustrated by a higher prevalence of infection and higher

parasitemia. The potential of the mosquito to serve as a vector depends on the ability to support

sporogony, mosquito abundance, and contact with humans, which are all influenced by climatic and

ecological factors. The ability to support sporogony is largely dependent upon species in that not all

species of anopheles are susceptible to plasmodium infection. Temperature and mosquito longevity are

other key factors affecting the parasite’s interaction with the vector. Development of Plasmodium

falciparum requires a minimum temperature of 200C, whereas the minimum temperature for other

species is 160C (Miller et al., 2002). Temperature also affects the time of development in that the

duration of sporogony is substantially shorter at higher temperature. A shorter duration of sporogony

increases the chances that the mosquito will transmit the infection within its lifespan.

Table 1: Factors influencing vectorial capacity

SPOROGONY MOSQUITO DENSITY HUMAN CONTACT

Temperature Temperature Anthropophilic

Mosquito longevity Altitude Indoor Vs Outdoor

Mosquito species Rainfall Feeding time

Breeding Places

(Miller et al., 2002)

Mosquito density and feeding habits also influence the transmission of malaria. Mosquito density is

affected by temperature, altitude, rainfall and availability of breeding places, whereas human-mosquito

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contact will be influenced by the mosquito behaviour. For example, the degree to which a

particular mosquito species is anthropophilic will influence the probability of the mosquito becoming

infected and then transmitting the infection to another human. These anthropophilic tendencies are

necessarily absolute in that many zoophilic mosquitoes will switch to humans if densities reach high

levels or the preferred animal source is diminished. The preferred feeding time and whether the

mosquito feeds predominantly indoors or outdoors will influence the transmission dynamics.

For example, outdoor feeding mosquitoes are likely to find human blood meal in early evening than

those feeding late at night when most people are inside. The behavior of the mosquito also needs to be

considered in controlling its activities.

1.2.7 Immunity Against Malaria

Persons living in endemic areas do develop immunity against malaria (Amador and Patarroyo, 1996).

Usually, a person will exhibit some symptoms during the initial exposure to malaria. Though symptoms

associated with subsequent exposures to malaria are usually less severe, the immunity against malaria

is slow to develop and requires multiple exposures. In highly endemic areas only young children are at

high risk of developing severe malaria, whereas older children and adults are essentially protected from

severe disease and death. However, this immunity is not a sterilizing immunity in that persons can still

become infected. In addition, the immunity is short-lived and in the absence of repeated exposure the

level of immunity decreases. For example, previously semi-immune adults will often develop severe

malaria upon returning to an endemic area after being in non-endemic area for 1–2 years. This state of

partial immunity in which parasitemia is lower, but not eliminated, and parasitemia is better tolerated is

sometimes referred to as premonition. Premonition refers to an immunity that is contingent upon the

pathogen being present.

The immune response could be directed at either the pre-erythrocytic or erythrocytic stage of the

parasite’s lifecycle (White, 1996). However, the erythrocytic stage of the lifecycle is probably the most

important in terms of clearing the parasites and lessening the disease. Possible effector mechanism for

antibody include blocking of the erythrocyte invasion by merozoites, antibody dependent cellular

killing mediated by cytophilic antibodies or increased clearance of the infected erythrocytes due to

binding of antibodies to parasite antigens exposed on the erythrocyte surfaces. All of these will result to

low parasitemia. The relative importance of these various mechanisms is not clear and probably

immunity requires the generation of antibodies against numerous targets. This, along with antigenic

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variation and polymorphisms in many plasmodium antigens, could explain the slow development

of immunity.

1.2.8 Human Genetics and Innate Resistance

Genetic resistance to malaria occurs through both modifications of the immune system that enhance

immunity to this infection and also by the changes in human red blood cells that hinder the malaria

parasite’s ability to invade and replicate within these cells (Mino and Gros, 2005). Host resistance to

malaria therefore involves not only the blood cell genes such as abnormal haemoglobins, glucose-6-

phosphate dehydrogenase deficiency and Duffy antigens which provide innate resistance but also

genes involved in immunity such as the major histocompatibility complex genes, which regulate

adaptive immune responses. The resistance provided by modified blood cells aids survival through the

dangerous years of early childhood, while the potent protection mediated by adaptive immune

responses is more important in older children and adults living where, malaria is endemic (Williams,

2006).

Certain genetic diseases and polymorphisms have been associated with decreased infection or disease

(Mino and Gros, 2005). For example, individuals who lack the Duffy blood – group antigen are

refractory to Plasmodium vivax. A large proportion of the populations in Western Africa are Duffy

negative, thus accounting for the low levels of Plasmodium vivax in West Africa. This innate resistance

led to the identification of the Duffy Antigens as the erythrocyte receptor for merozoite invasion

(Williams, 2006).

Several inherited erythrocyte disorders are found predominantly in malaria endemic areas and at

frequencies much higher than expected. This has led to speculation that these disorders confer some

protection against malaria (Mino and Gros, 2005). The combination of defect and infection lead to

premature lysis or clearance of the infected erythrocyte. For example, glucose-6-phosphate

dehydrogenase (G6PD) deficient erythrocytes would have an impaired ability to handle oxidative

stress. Then, the additional oxidants produced as a result of parasite metabolism and the digestion of

hemoglobin may overwhelm the infected erythrocyte and lead to its destruction before the parasite is

able to complete schizogony. Sickle cell anemia and thalassemia are also speculated to make the

infected erythrocyte more susceptible to oxidative stress (Williams, 2006).

1.2.9 Diagnosis of Malaria

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Malaria is suspected in persons with a history of being in an endemic area and presenting

symptoms consistent with malaria (Beare et al., 2006). The mainstay of malaria diagnosis has been

microscopic examination of the blood. Although, blood is the sample most frequently used to make

diagnosis, both saliva and urine have been investigated as alternative less invasive specimen. Areas that

cannot afford laboratory diagnostic tests often use only history of subjective fever as the indication to

treat for malaria (Beare et al., 2006). Using giemsa-stained blood smears from children in Malawi were

adopted instead of clinical prediction as treatment indications, rather than using only a history of

subjective fevers, a correct diagnosis increased from 2% to 41% of cases, and unnecessary treatment

for malaria was significantly decreased. Some of the methods of diagnosis are :

1.1.9.1 Blood Films

The most economic, preferred and reliable diagnosis of malaria is the microscopic examination of

blood films because each of the plasmodium species have distinguishing parasitic characteristics (Beare

et al., 2006). Two type of blood films are used traditionally. Thin films are usually the most used and

allow species identification because the parasite’s appearance is best preserved in this preparation

(Redds et al., 2006). Thick films allow the microscopist to screen a larger volume of blood and are

about eleven times more sensitive than the thin film, so picking up low levels of infection is easier on

the thick film, but the appearance of the parasite is very difficult to detect. But, it is usually imperative

to utilize the two types of smears while attempting to make a definitive diagnosis (Redds et al., 2006).

For areas where microscopy is not available or where laboratory staff are not experienced at malaria

diagnosis, there are commercial antigen detection tests that require only a drop of blood (Warhust and

Williams, 1996). Immunochromatographic tests also called Malaria Rapid Diagnostic Tests, Antigen-

capture Assay or Dipsticks have been developed, distributed and Field-Tested. These tests use finger-

stick or venous blood, the complete test takes a total of 15 – 20 minutes, and the results are read

visually as the presence or absence of coloured stripes on the dipstick, so they are suitable for use in the

field. The thresholds of detection by these rapid diagnostic tests are in the range of 100 parasite/µl of

blood. The disadvantage is that dipstick tests are qualitative but not quantitative they can determine if

parasites are present in the blood, but not how many.

1.1.9.2 Molecular Methods

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Molecular methods are available in some clinical laboratories and rapid real-time assays. for

example, QT-NASBA, based on the polymerase chain reaction (PCR), are being developed with the

hope of being able to deploy them in endemic areas. PCR and other molecular methods are more

accurate than those based on microscopy (Mc Cutchan et al., 2008). However, it is expensive and

requires a specialized laboratory. Moreover, levels of parasitemia are not necessarily correlative with

the progression of disease, particularly when the parasite is able to adhere to blood vessel walls.

Therefore, more sensitive low cost diagnostic tools need to be developed in order to detect low levels

of parasitemia in the field (Mc Cutchan et al., 2008).

1.2.10 Prevention and Control of Malaria

Strategies for preventing and controlling malaria involve three different approaches which include:

Reduction of human-mosquito contacts

Reduction of the vector density and

Reduction of parasite reservoir (Phillips, 2001).

Prevention of malaria in individuals will generally involve the reduction of human-mosquito contacts

through the use of bednets, repellents and house spraying. Also, chemoprophylaxis can be used

especially in travelers. Chemoprophylaxis only suppresses parasitemia but does not prevent infection.

Controlling activities at the community level can utilize approaches which directly reduce human-

mosquito contact as well as, approaches which reduce the total number of mosquitoes in an area. Such

approaches include the reduction in mosquito breeding grounds; target the larvae stages with chemical

or biological agents and massive insecticide spraying for the adult mosquitoes. Biological control

methods include the introduction of fish which eat the mosquito larvae, for example Bacillus

thuringiensis which excrete larval toxins. Case detection and treatment is another potential control

method. Identifying and treating infected persons, especially asymptomatic individuals will reduce the

size of the parasite reservoir within the human population and can be a relatively expensive approach.

These approaches are not mutually exclusive and can be combined. Many of the successful control

programmes include both measures to control mosquitoes and treatment of infected individuals

(Phillips, 2001). There is no standard method of malaria control that has proven universally effective.

The epidemiology, socio-economic, cultural and infrastructural factors of a particular region will

determine the most appropriate malaria control. Some of the factors which need to be considered are:

Infrastructure of existing healthcare service and other resources

Intensity and periodicity of transmission

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Mosquito species

Parasite species and drug sensitivities

Cultural and social characteristics of the population

Presence of social and ecological changes.

The control of malaria in tropical Africa has been particularly problematic because of high transmission

rates and the overall low socio-economic level. Several studies have shown that insecticide treated

bednets (ITBN) reduce the morbidity and mortality associated with malaria (Pasvol, 2006). In most

areas the introduction of bednets do not require large promotional programmes and their uses are

readily accepted (Pasvol, 2006). This may be partly due to the reduction in mosquito irritating biting. It

is necessary to retreat the bednets with insecticide periodically and the bednets need to be repaired and

replaced as they become torn and worn out. In addition, some have raised concerns about the long-term

benefits of bednets since they reduce exposure, but do not eliminate it. This reduction in exposure may

delay the acquisition of immunity and simply postpone morbidity and mortality to older age groups.

1.2.11 Anti-Malarial Drugs

Drugs which are used for prophylaxis, treatment and in the prevention of malaria are called anti-

malarials. These drugs could be used in the

Treatment of malaria in individuals with suspected or confirmed infection

Prevention of infection in individuals visiting a malaria-endemic region, who have no immunity

Routine intermittent treatment of certain groups in endemic regions

Hence, some agents are used for more than one application. It is therefore, more practical to group anti-

malaria agents by their chemical structure since this is associated with their drug properties, such as

mechanism of action (White, 2004).

Several anti-malarial drugs are available. Many factors are involved in deciding the best treatment for

malaria. These factors include:

The parasite species

The severity of disease (complicated)

The patience’s age and immune status

The parasite’s susceptibility to the drugs (drug resistance) and

The cost and availability of drugs.

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Therefore, the exact recommendations will often vary according to the geographical region. So,

various drugs act differently on the different lifecycle stages (Pasvol, 2006).

Table 2: Selected anti-malarial drugs

S/N DRUG CLASS EXAMPLE

1. Fast-acting blood

schizontocide

Chloroquine (+ other 4-aminoquinolines), quinine, quinidine,

mefloquine, halo-flantrine, antifolates (Pyrimethamine,

proquanil, sulfadoxine, dapsone).

2. Slow-acting blood

schizontocide

Doxycycline (+ other tetracycline antibiotics)

3. Blood + Mild tissue

schizontocide

Proquanil, pyrimethamine, tetracyclines

4. Tissue schizontocide

(anti-relasping)

Primaquine

5. Gametocidal Primaquine, artemisinin derivative, 4-aminoquinolines

6. Combinations Fansidar (primethamine + sulfadoxine), maloprim

(pyrimethamine + dapsone), malarone (atovaquone + proquanil)

(Pasvol, 2006)

Fast-acting blood schizontocides, which act upon the blood stage of the parasite, are used to treat acute

infections and to quickly relieve the clinical symptoms. Chloroquine is generally the recommended

treatment for patient with Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and

uncomplicated chloroquine- sensitive Plasmodium falciparum infections. Chloroquine is safe and

usually well tolerated. Side effects may include itching, nausea, or agitation. Patients infected with

either Plasmodium vivax and Plasmodium ovale that are not at a high risk for reinfection, should be

treated with primaquine (a tissue schizontocide). Primaquine is effective against the liver stage of the

parasite, including hypnozoites and will prevent future relapses. The combination of chloroquine and

primaquine is often called radical cure (Pasvol, 2006). Severe or complicated, falciparum malaria is a

serious disease with a high mortality rate and must be regarded as life threatening and thus requires

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urgent treatment. Treatment typically requires parenteral drug administration (i.e. injections) since

the patients is often vomiting and thus cannot take the drugs orally. Parenteral formulations are equally

available for chloroquine, quinine, quinidine and artemisinin derivatives. The artemisinin derivatives

are generally the preferred choice, but are not yet approved. For example, in the United States, quinine

and quinidine are the approved drugs for severe malaria (White, 2008). Patients are screened of

parasitemia, hydration levels, hypoglycemia and signs of drug toxicity and other complication during

the course of treatment. Most deaths due to severe malaria occur at or close to home in situations where

the patients cannot be taken to the hospital. Artemisinin suppositories which can be administered by

village health workers have also been developed and have proved to be safe and effective (White,

2008).

The efficacy of chloroquine is greatly diminished by the widespread of chloroquine resistant

Plasmodium falciparum and also, the emergence of chloroquine resistant Plasmodium vivax. In an area

with chloroquine resistant malaria, the common alternatives include the use of mefloquine, quinine in

combination with doxycycline, fansidar, derivatives of artemisinin (dihydroartemisinin, artesunate and

artemether) are increasingly used in Asia and Africa. It is now recommended as the first line of

treatment by the World Health Organization. These drugs were originally derived from wormwood

plant (Artemesia annua) and have been used for a long time in China as an herbal tea called quinhaosu

to treat febrile illnesses. To prevent high recrudescence and to slow the development of drug resistance,

it is recommended that the treatment will be combined with an un-related anti-malarial (Ogwal, 1996).

Drugs used in combination with artemisinin include mefloquine, lumefantrine, fansidar and

amodiquine.

1.1.11.1 Chemoprophylaxis

Chemoprophylaxis is particularly important for persons from non-endemic areas who visit areas

endemic for malaria (White, 1996). Such non-immune persons can quickly develop a serious and life-

threatening disease. As in the case of treatment there is no standard recommendation and the choices

for chemoprophylaxis are highly dependent upon the individual concerned (Newton and White, 1999).

Chemoprophylaxis drugs should be non-toxic since these drugs will be taken over an extended periods

of time.

1.1.12 Drug Resistance

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Anti-malarial drugs resistance has been defined as the ability of a parasite to survive or multiply

despite the administration and absorption of a drug given in doses equal to or higher than those usually

recommended but within the tolerance of the subject. The drug in question must gain access to the

parasite or the infected red blood cell for the duration of the time necessary for its normal action. In

most instances, this refers to parasites that remain from an observed treatment. In order for a case to be

defined as resistant, the patient must have received a known and an observed anti-malarial therapy

whilst the blood and metabolite concentrations are monitored concurrently. The techniques used to

demonstrate this are in vivo, in vitro animal model testing and the most recently developed molecular

techniques.

Drug resistant parasites are often used to explain malaria treatment failure (Boland, 2001). However,

there are two potentially very different clinical scenarios. The failure to clear parasitemia and recover

from an acute clinical episode when a suitable treatment has been given, then anti-malarial resistance is

in its true form. Drug resistance may lead to treatment failure, but treatment failure is not necessarily

caused by drug resistance despite assisting to its development (Warhust, 2001). A multitude of factors

can be involved in the processes including problems with non-compliance and adherence, poor drug

quality, interactions with other pharmaceuticals, poor absorption, misdiagnosis and incorrect doses

being given. The majority of these factors also contribute to the development of drug resistance.

The development of resistance can be complicated and varies between plasmodium species as follows:

It is generally accepted to be initiated primarily through a spontaneous mutation that provides

some evolutionary benefit, thus giving an anti-malaria used a reduced level of sensitivity

This can be caused by a single point mutation or multiple mutations. In most instances a

mutation will be fatal for the parasite however, some resistant parasites will survive. Resistance

can become firmly established within a parasite population, existing for long periods of time

(Hyde, 2007). The first type of resistance to be recognised was to chloroquine in Thailand in

1957. The biological mechanism behind this resistance was subsequently discovered to be

related to the development of an efflux mechanism that expels chloroquine from the parasite

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before the level required to effectively inhibit the process of haem polymerization (i.e.

necessary to prevent build up of the toxic byproducts formed by haemoglobin digestion)

(Hyde, 2007). This theory has been supported by evidence showing that resistance can be

effectively reversed on the addition of substances which can halt the efflux.

The resistance of other quinolone anti-malarials such as mefloquine, halofantine and quinine are

thought to have occurred by similar mechanisms (Hyde, 2007). Also, plasmodium have developed

resistance against antifoliate combination drugs, the most commonly used being sulfadoxine and

pyrimethamine. Two gene mutations are thought to be responsible, allowing synergistic blockages of

two enzymes involved in foliate synthesis. Regional variations of specific mutations give differing

levels of resistance.

Atovaquone is recommended to be used only in combination with another anti-malarial compound as

the selection of resistant parasites occurs very quickly when used in monotherapy. Resistance is

thought to originate from a single point mutation in the gene coding for cytochrome b.

1.1.12.1 Spread of Resistance

There is no single factor that confers the greatest degree of influence on the spread of drug resistance .

A number of plausible causes associated with an increase have been advocated. These include aspects

of economics, human behaviour, pharmacokinetics and the biology of vectors and parasites.

The most influential causes of spread of resistance are listed below:

The biological influences are based on the parasites ability to survive the presence of anti-

malarial, thus, enabling the persistence of resistance and the potential for further

transmission despite treatment. In the normal circumstances any parasite that persist after

treatment are destroyed by the host’s immune system. Therefore any factors that act to

reduce the elimination of parasites could facilitate the development of resistance. This

explains the poorer response associated with immunocompromised individuals, pregnant

women and young children.

There has been evidence that certain parasite- vector combinations can alternatively enhance or inhibit

the transmission of resistant parasites.

The use of antimalarials developed from similar basic chemical compounds can increase

the rate of emergence of resistance for example, cross-resistance to chloroquine and

aminodiaquine, two 4-aminoquinolones and mefloquine conferring resistance to quinine

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and halofantrine. This phenomenon may reduce the usefulness of newly developed

therapies prior to large scale usage.

The resistance to anti-malarials may be increased by a process found in some species of

plasmodium, where a degree of phenotypic plasticity was exhibited, allowing the rapid

development of resistance to a new drug, even if the drug has not been previously used.

The pharmakinetics of the chosen anti-malarial are keys; the decision of choosing a long

half-life over a drug that is metabolized quickly is complex and still remains unclear. Drugs

with shorter half-life require more frequent administration to maintain the correct plasma

concentrations. Longer-lasting drugs can increase the development of resistance due to

prolonged periods of low drug concentration.

The pharmakinetics of anti-malarials are important when using combination therapy,

mismatched drug combination, for example, having an unprotected period when one drug

dominates can seriously increase the likelihood of selection for resistant parasites.

Individuals may only take the drugs until symptoms clear or will take lower doses to save

money

Individuals may not complete the full course of treatment because of drug side effects.

The widespread use of a drug in an area of intense transmission increases drug pressure by

exposing a larger parasite population to the drug.

High levels of transmission may allow re-infection while drugs are at sub-therapeutic

levels.

1.1.12.2 Prevention of Resistance

The prevention of anti-malarial drug resistance is of enormous public health importance (Wellem and

Plowe, 2001). It can be assumed that no therapy currently under development or to be developed in the

foreseeable future will totally be protective against malaria. In accordance with this, there is a

possibility of resistance emerging for any given therapy that is developed. This is a serious concern, as

the rate at which new drugs are produced by no means matches the rate of the development of

resistance. In addition, the most newly developed therapeutics tend to be the most expensive and are

required in the largest quantities by some of the poorest areas of the world.

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Therefore, it is apparent that the degree to which malaria can be controlled depends on the careful

use of the current drugs to limit any further development of resistance. Provisions essential to this

process include the delivery of fast primary healthcare where staff are well trained and supported with

the necessary supplies for efficient treatment.

Preventing malaria has a substantial effect on the potential rate of development of resistance, by

directly reducing the number of cases of malaria thus decreasing the requirement for anti-malarial

therapy (Wellems, 2002). So, by preventing the transmission of resistant parasites limits the risk of

resistant malarial infections becoming endemic and can be controlled by a variety of non-medical

methods including insecticide-treated bednets, indoor residual spraying, environmental controls (such

as swamp draining) and personal protective methods such as using mosquito repellent.

Chemoprophylaxis is also important in the transmission of malaria infection and the resistance in

defined populations e.g. travelers (Wellems and Plowe, 2001).

A hope for future of anti-malarial therapy is the development of an effective malaria vaccine

(Wongsrichanalai et al., 2002). This could have enormous public health benefits, providing a cost

effective and easily applicable approach to preventing not only the onset of malaria but the

transmission of gametocytes, thus reducing the risk of resistance development.

1.3 MORINGA OLEIFERA

Moringa oleifera, commonly referred to as Moringa is the most widely cultivated species of the genus

Moringa, which is the only genus in the family of Moringaceae. It is an exceptionally nutritious

vegetable tree with a variety of potential uses (Caceres et al., 1991). The tree itself is rather slender,

with dropping branches that grow to appropriately 10m in height. In cultivation, it is often cut back

annually to 1 meter or less and allowed to re grow so that pods and leaves remain within arm’s reach.

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Fig. 1: Pictorial view of Moringa oleifera

Table 3: Scientific classification of Moringa oleifera

KINGDOM PLANTAE

Unranked Angiosperms

Unranked Eudicots

Unranked Rosids

Order Brassicales

Family Moringaceae

Genus Moringa

Species Moringa oleifera

(Anwar et al., 2007)

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1.2.1 Distribution of Moringa oleifera

The Moringa tree is grown mainly in semi-arid tropical and subtropical areas. It grows best in dry

sandy soil. It is a fast- growing drought-resistant tree that is native to the Southern foothills of

Himalayans in northwestern India. It is considered as one of the world’s most useful trees, as almost

every part of the Moringa tree can be used for food or has some other beneficial properties (Anamika et

al ., 2010). In the tropics, it is used as forage for livestock and in many countries as vegetables that has

the potential to improve nutrition, boost food security, and foster rural development and support

sustainable land care.

1.2.3 General Nutrition of Moringa oleifera

The immature green pods called drumsticks are probably the most valued and widely used part of the

tree. They are commonly consumed in India and are generally prepared in a similar fashion to green

beans and have a slight asparagus taste (Foidl et al., 2001). The seeds are sometimes removed from

more mature pods and eaten like peas or roasted like nuts. The flowers are edible when cooked and are

said to taste like mushrooms. The roots are shredded and used as a condiment in the same way as

horseradish.

The leaves are highly nutritious, being a significant source of beta-carotene, vitamin C, protein, iron

and potassium (Makkar and Becker, 1997). The leaves are cooked and used like spinach. In addition to

being used as a substitute for spinach, its leaves are commonly dried and crushed into a powder and

used in soups and sauces. The tree is also a good source of calcium (Makkar and Becker, 1997). In

Siddha medicines, these drumstick seeds are used as a sexual virility drug for treating erectile

dysfunction in men and also in women for prolonging sexual activity. The Moringa seeds yield 38-40%

edible oils. The refined oil is clear, odourless and resists rancidity better than other oil. The seed cake

remaining after oil extraction may be used as a fertilizer or as a flocculent to purify water . The bark,

sap, roots, leaves, seeds oil and flowers are used in traditional medicine in several countries.

Moringa oleifera, grown and used in many countries around the world, is a multi-purpose tree with

medicinal, nutritional and socio-economical values (Bodeker and Willcox, 2000). In Senegal and

Benin, Moringa oleifera leaves are dispensed as powder at health facilities to treat malnutrition in

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children (Willcox et al., 2005). It was massively grown and promoted by the local media in

Uganda in the 1980s as a plant putatively able to cure a number of diseases including malaria and

symptoms of HIV/AIDS (Willcox, 1999).

1.3 AIM AND OBJECTIVES OF THE STUDY

The aim of this study was to investigate the effectiveness of Moringa oleifera ethanol leaf extract in the

treatment of malaria.

The specific objectives were as follow:

To determine the acute toxicity (LD50) and phytochemical constituents in Moringa oleifera

ethanol leaf extract.

To determine the percentage parasitaemia in mice and the effect of Moringa oleifera ethanol

leaf extract on the percentage parasitaemia within the pre- and post-treatment periods.

To determine the effect of Moringa oleifera ethanol leaf extract on haematological parameters

in malaria-induced mice within the pre- and post-treatment periods.

To determine the effect of Moringa oleifera ethanol leaf extract on the liver marker enzymes in

malaria-induced mice within the post-treatment periods.

To determine the effect of Moringa oleifera ethanol leaf extract on some kidney markers in

malaria-induced mice within the post-treatment periods.

To determine the effect of Moringa oleifera ethanol leaf extract on lipid profile in malaria-

induced mice within the post-treatment periods.

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Animals

The experimental animals used for this study were white albino mice of either sex weighing 20 – 34g.

The mice were between 3 – 4 months old and were obtained from the animal unit of Faculty of

Veterinary Medicine, University of Nigeria, Nsukka.

2.1.3 Moringa oleifera (Agbaji) Leaves

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Moringa oleifera (Agbaji) leaves were plucked from Moringa oleifera trees in various locations in

Ovoko, Igbo-Eze South L.G.A of Enugu Sate, Nigeria. The leaves were identified by Mr. O. Chijioke

of the Hebarium Unit of the Department of Botany, University of Nigeria , Nsukka.

2.1.4 Instruments/Equipment

Water bath (Gallenkamp, England)

Chemical balance (Gallenkamp, England)

Test tubes (Pyrex, England)

Conical flask (Pyrex, England)

Hot box (Gallenkamp, England)

Centrifuge (Pic, England)

Syringe & needle(1ml and 5ml) (Dana Jet, Nigeria)

Microscope slides (Unescope, USA)

Digital photo colorimeter (E1,312 Model, Japan)

Adjustable micropipette (Perfect, USA)

Refrigerator (Kelvinator, Germany)

pH meter (Pye, Unicam 293, England)

Stirrer (Sward, England)

Capillary tubes (Pyrex, England)

2.1.5 Chemicals/Reagents

All the chemicals used in this study were of analytical grade and products of May and Baker, England;

BDH, England and Merck, Darmstadt, Germany. The reagents used for all the assays were commercial

kits and products of Randox, QCA, USA and biosystem Reagents and Instruments, Spain.

2.2 METHODS

2.2.1 Extraction

The leaves of Moringa oleifera plant were plucked and then dried under room temperature at (290C -

350C) for three weeks, after which the leaves were pulverized into coarse form with a Crestor high

speed milling machine. The coarse form (1kg) was then macerated in 5 volume (w:v) absolute ethanol.

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This was left to stand for 48 hours. After that the extract was filtered through muslin cloth on a

plug of glass wool in a glass column. The resulting ethanol extract was concentrated and evaporated to

dryness using rotary evaporator at an optimum temperature of between 40 and 450C to avoid

denaturation of the active ingredients. The concentrated extract was stored in the refrigerator for

subsequent studies.

2.2.2 Experimental Design

Twenty-four white albino mice of either sex weighing 20 – 34kg were housed in separate cages,

acclimatized for one week and then divided into six groups of four mice each. The route of

administration (treatment) was via oral route with the aid of an oral intubation tube.

Group 1 was the (positive control) inoculated with malaria parasite (Mp+) and treated with 5mg/kg

body weight of distilled water.

Group II was inoculated with malaria parasite and treated with 45mg/kg body weight of Moringa

oleifera ethanol leaf extract.

Group III was also inoculated with malaria parasite and treated with 90mg/kg body weight of Moringa

oleifera ethanol leaf extract.

Group IV was inoculated with malaria parasite and treated with 180mg/kg body weight of Moringa

oleifera ethanol leaf extract.

Group V which was also inoculated with malaria parasite (standard control) and was treated with

5mg/kg body weight of artesunate (standard drug).

Group VI was the negative control which was not inoculated with malaria parasite and was finally

treated with 5mg/kg body weight of distilled water.

Before the treatments, the mice in Groups I – V were inoculated with malaria parasite and 3 days after

that analyses were carried out to determine the baseline parameter in all the groups, then, two days

later, treatment began. The treatment lasted for 5 days during which analyses were done on day 3, day

5 of treatment and 28 days post treatment .

Several parameters were investigated using whole blood. The parameters were

Packed Cell Volume (PCV)

Malaria parasite test

Total White Blood Cell Counts (TWBC)

Total Red Blood Cell Counts (TRBC)

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Haemoglobin Tests (Hb)

Parameters studied using serum were

Alanine aminotransferase activity (ALT)

Aspartate aminotransferase activity (AST)

Alkaline phosphatase activity (ALP)

Total Bilirubin concentration (TB)

Serum Creatinine concentration

Urea Concentration concentration

Total Serum Cholesterol concentration

Triacyglycerol concentration

High Density Lipropotein (HDL) concentration

Low Density Lipoprotein (LDL) concentration

2.2.3 Procurement of Parasitaemia

Malaria parasite (Plasmodium berghei) was obtained from malaria infected- mice at the Department of

Veterinary Medicine, University of Nigeria, Nsukka. Ten drops of the parasitized blood obtained with

the aid of a capillary tube through the ocular region of the mice, were diluted with 1 ml of normal

saline. Thereafter, 0.2 ml of the diluted parasitized blood was used to infect the three mice that served

as the host from where other experimental animals were infected.

2.2.5 Preparation of EDTA (Sequestrene) Anticoagulant

EDTA anticoagulant was prepared by dissolving 2.5g of di-potassium ethylene in 25ml of distilled

water. The bottle was labeled and 0.04 ml of the anticoagulant reagent was pipetted into bottles marked

to hold 2.5 ml of blood. The small bottles ,protected from dust and flies, were placed without tops on a

warm bench for the anticoagulant to dry. Then, when dried, the bottle were capped and stored in a

refrigerator ready to be used.

2.2.6 Preparation of Giemsa Stain

Exactly 3.8g of Giemsa powder was transferred to a dry brown bottle of 500 ml capacity. With the aid

of a dry measuring cylinder, 250ml of methanol was measured and added to the Giemsa powder and

mixed well; 250ml of glycerol was also added to the stain and stirred very well. Then, the bottle

143

containing the stain was placed in a water bath at 370C for up to 2 hours to help the stain dissolve.

The mixture was stirred at intervals. The bottle was labelled and marked flammable and toxic. It was

kept at room temperature ready for use.

2.2.6 Preparation of Alcohol Fixative Solution

Exactly 180 ml of absolute ethanol was added into 250ml cylinder capacity. This was followed by

addition of 10 ml of distilled water and 10ml of glacial acetic acid into 200ml marked container and

mixed. The bottle was labelled flammable and ready for use.

2.2.7 Methods of Estimations.

2.2.7.1 Determination of Malaria Parasitaemia

The determination of malaria parasitemia (Mp+) was carried out according to the Method of Dacie and

Lewis (2000). A swab moistened with 70% v/v alcohol was used to cleanse the tail of a mouse and

allowed to dry. A pair of scissors was used to cut the tail which was squeezed gently to obtain a small

drop of blood that was placed on the centre of a microscope slide. Immediately the thin film was spread

using a smooth edged slide spreader. The slide was labeled with a black lead pencil and air-dried in

horizontal position.

i. Fixation of the thin blood films: The slide was horizontally placed on a level staining rack. A

small drop of absolute ethanol was applied to the thin film, using a swab. This was allowed to

fix for 2 minutes.

ii. Giemsa Staining Technique: A volume of 50 ml of buffered saline pH 7.1 – 7.2 was added to

1.5ml of Geimsa stain and mixed gently. The slides were placed face downwards in a shallow

tray supported on two rods in a staining rack. Then, the diluted stain was poured into the

shallow tray and allowed for 30 minutes, after which the stain was washed off from the staining

container using clean water. Finally, the back of the slide was wiped and placed in a draining

rack for air-drying. With the aid of counting chamber, dried stained film was viewed

microscopically using 100 x objective.

iii. Counting the Percentage of Parasitized Red Cells: 100 x objective was used to select an area

of the thin film where the total number of red cells was approximately 250 per field. The

numbers of parasitized red cells were counted in 8 fields, which were approximately 2000 cells.

Then, the number of parasitized red cells was divided by 20 to get the percentage of parasitized

cells.

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% Parasitized = 20

cells red dparasitize ofNumber

Blood samples that were used for haematological analyses were collected with the help of a

capillary tube via the ocular region of the mice and placed in an EDTA tube.

2.2.7.2 Determination of Total Red Blood Cell Count

The determination of total red blood cell count was carried out according to the method of Dacie and

Lewis (2000).

Principle

The blood specimen was diluted 1:200 with the red blood cell diluting fluid and cells were counted

under high power (40 X objective) by using a counting chamber. The number of cells in the blood were

calculated and reported as the number of red cells per µl of the whole blood.

Methodology

Blood from EDTA tube was mixed carefully by swirling the bulb. The blood was drawn quickly

with red blood cell pipette up to the 0.5 mark, then, excess blood outside the pipette was carefully

wiped using cotton. This was equally used to draw diluting fluid up to the 101 mark. The pipette was

rotated rapidly by keeping it horizontally during mixing. The cell was allowed to settle 2 to 3 minutes,

then, counting chamber was placed on the stage of the microscope. The microscope was switched to

low power (10x) objective. Its light was adjusted to locate the large square in the centre with 2 small

squares. Then, the microscope was switched to high power (40x) objective. Finally, the red blood cells

in the four corner squares and in the centre square were counted.

Total red blood cells per litre of blood =10 Dilution Counted Volume

Counted Cell ofNumber

2.2.7.17 Determination of Total White Blood Cell Count

The determination of total white blood cell count was carried out according to the method of Dacie and

Lewis (2000).

Principle

145

Whole blood was diluted 1 in 20 in an acidic reagent which haemolyzes the red cells, leaving the

white cells to be counted. White cells are counted microscopically using a counting chamber. .

Methodology

The counting chamber and cover slip were cleaned with water, dried and mounted on the mechanical

stage of the microscope. The blood sample was pipetted from the EDTA tube up to the 0.5 mark of the

pipette, followed by drawing of the diluting fluid up to the 11 mark from the watch glass by keeping

the pipette in a vertical position. The blood and diluting fluid were allowed to mix well by rolling the

pipette horizontally in between the palms. The cells were allowed to settle for 2 to 3 minutes. The cells

were counted microscopically by using 10 x objectives. This was used to focus the four large corner

squares of the chamber. The number of white cells per litre of blood was calculated using the

following formula:

WBC = 20

counted cell ofnumber Total

The number obtained which was multiplied by a factor of 109 gives the white cell count.

2.2.7.18 Determination of Packed Cell Volume (PCV)

Packed cell volume (PCV) was determined by the method of Dacie and Lewis (2000).

Principle

Anticoagulated blood in a glass capillary of specified length, bore size and the wall-thickness is

centrifuged in a micro-haematocrit centrifuge at RCF 10,000-11,000 rpm for 5 minutes to obtain

constant packing of the red cells. A small amount of plasma remains trapped between the packed red

cells. The PCV value is read from the scale of a micro-haematocrit reader or calculated by dividing the

height of the red cell column by the height of the total column of the blood.

Methodology

A heparinized capillary tube was filled with blood from an EDTA tube up to the three quarters of the

capillary tube. The end of the tube was sealed with a plasticine sealant. The capillary tubes were

arranged according to their number in the micro-haematocrit, then, followed by centrifuging for 5

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minutes (RCF 10,000-11,000 rpm). Immediately after centrifuging, the PCV was read by using a

micro-haematocrit reader by aligning the base of the red cell column above the sealant on the 0 line and

the top of the plasma column on the 100 line.

PCV = (mm) Total ofLength

(mm)Count Cell Red ofLength

2.2.7.19 Determination of Haemoglobin (Hb) Concentration

Haemoglobin (Hb) concentration was determined using haemoglobincyanide (HICN) technique as

outlined in the method of Dacie & Lewis (2000).

Principle

Whole blood was diluted 1 in 201 in a modified Drabkin’s solution which contains potassium

ferricyanide and potassium cyanide. The red cells are haemolyzed and the haemoglobin is oxidized by

the ferricyanide to methaemoglobin. This was converted by cyanide to stable haemiglobincyanide

(HiCN). The absorbance of the HiCN solution was read at wavelength of 540nm. The absorbance

obtained was compared with that of a reference HiCN standard solution. Haemoglobin values are

obtained from tables prepared from a calibration graph.

Methodology

A volume of 20µl of the capillary blood was dispensed into 4 ml of Drabkin’s neutral diluting fluid in a

tube. The tube was stoppered, mixed and left at room temperature (29–35oC) for 5 minutes. The

colorimeter was adjusted to 540 nm followed by zeroing it with Drabkin’s fluid and reading of the

absorbance of the sample. With the aid of table prepared from the calibration graph, the mice

haemoglobin values were read.

Concentration of HiCN in 1000

200/ lmg

Preparation of a calibration curve.

Six tubes were taken and labelled blank B, 1, 2, 3, 4 and 5. The following reagents were pipetted into

the test tubes as follows

Tests B 1 2 3 4 5

1ml in 20ml diluted standard - 4 3 2 1 5

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1 ml of ammonia water 5 1 2 3 4 -

The colorimeter was adjusted to 540nm followed by zeroing with Drabkin’s neutral fluid in B

and reading of the absorbance. The haemoglobin (Hb) equivalent in g/l of solution in tubes 1- 5 was

calculated as follow:-

Tube 1 Hb value of HiCN standard x = Hb g/l

Tube 2 Hb value of HiCN standard x = Hb g/l

Tube 3 Hb value of HiCN standard x = Hb g/l

Tube 4 Hb value of HiCN standard x =Hb g/l

Tube 5 Hb value of HiCN standard = Hb g/l

A graph of absorbance against concentration was plotted for Hb with values from 20- 200g/l or 2 –

20g/dl .

2.2.7.20 Determination of Total Bilirutin Concentration

Total bilirubin concentration was determined using the method of Jendrassik and Grof (1938) as

outlined in the Randox kit.

Principle

Direct (conjugated) bilirubin reacts with diazotized sulphanilic acid in alkaline medium to form a blue

coloured complex. Total bilirubin is determinded in the presence of caffeine, which releases albumin

bound bilirubin by reaction with diazotized sulphanilic acid.

Methodology

A volume of 0.2 ml of sulphanilic acid was pipetted into the sample blank tube and sample tube. This

was immediately followed by the addition of 0.05 ml of sodium nitrite to the sample tube. Caffeine

(10ml) and 0.2 ml of sample were also pipetted into each of the sample blank tube and sample tube.

These mixtures were mixed and incubated for 10 minutes at 20 -250C. Finally, 1.0 ml of tartrate was

148

pipetted into the sample blank tube and sample tube. These mixtures were once again mixed,

incubated for 30 minutes at 20 – 250C and their absorbances read at 578nm against the sample blank.

Total bilirubin (µmol/l) = 185 × sample blank (578nm)

2.2.7.21 Determination of Serum Urea Concentration

The concentration of serum urea was determined using the method of Tietz (1994) as outlined in

Randox kits, UK.

Principle

Urea in serum is hydrolysed to ammonia and is then measured photometrically .

Urea + H2O Urease

2NH3 + CO2

NH3 + Hypochlorite + Phenol Indophenol

Methodology

A known volume, 10µl of the sample was pipetted into the sample tube, 10µl of the standard was also

pipetted into the standard tube followed by addition of 10µl of distilled water to the blank tube. A

volume of 10µl sodium nitroprusside and urease were added to each of the three tubes. The tubes were

mixed and incubated at 370C for 15 minutes. Then, 2.50 ml of phenol was added to each of the three

tubes followed by addition of 2.50 ml of sodium hypochlorite also. These were mixed and incubated

for 15 minutes at 370C and the absorbance was read against the reagent blank at 546nm.

Urea concentration (mmol/l) = Standard

Sample

A

A× Standard concentration

ASample = Absorbance of sample

AStandard = Absorbance of standard

2.2.7.22 Determination of Serum Creatinine Concentration

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The concentration of serum creatinine was determined using the method of Tietz (1994) as

outlined in Randox kits, UK.

Principle

Creatinine in alkaline solution reacts with picric acid to form a coloured complex. The amount

of the complex formed is directly proportional to the creatinine concentration. A known volume, 100 µl

of distilled water was pipetted into the blank tube; also 100 µl of the standard was pipetted to the

standard tube while 100 µl of the sample was pipetted into the sample tube. Then, 100 µl of the

working reagents were pipetted into the three tubes and mixed. The absorbance of the sample was read

against the blank at 492 nm.

Creatinine (µmol/l) = Standard

Sample

A

A x Standard Concentration

ΔASample = Change in Absorbance of sample

ΔAStandard = Change in Absorbance of standard

2.2.7.23 Assay of Aspartate Aminotransferase (AST) Activity

The activity of aspartate aminotransferase was assayed by the method of Reitman and Frankel (1957)

as outline in Randox kit.

Principle

Oxaloacetate is formed according to the equation:

α – Oxoglutarate + L-aspartate AST

L-glutamate+ Oxaloacetate

Methodology

Aspartate aminotransferase activity was assayed by monitoring the following information of

oxaloacetate hydrazone with 2, 4-dinitrophenylhydrazine.

Kit Reagents

150

Mea

sure

men

t

Aga

inst Reagent Blank

The AST substrate phosphate buffer of 0.5ml each was pipetted into both the reagent blank (B) and

sample test (T) test tubes respectively. The serum sample of 0.1 ml was added to the sample test (T)

test tubes only and mixed thoroughly. Then, 0.1 ml of distilled water was added to the reagent blank

(B). Then, the entire reaction medium was well mixed and incubated for 30 minutes in a water bath at

370C.

Immediately after incubation, 2, 4-dinitrophenylhydrazine (0.5 ml) was added to the reagent blank (B)

and the sample test tubes. It was mixed thoroughly and allowed to stand for exactly 20 minutes at 250C.

Finally, 5.0 ml of sodium hydroxide solution was added to both the blank and the reagent and the

reagent test tubes respectively and mixed thoroughly. The absorbance of sample Asample was read at a

wavelength of 550nm against the reagent blank after 5 minutes.

Measurement Against Sample Blank

The AST substrate phosphate buffer of 0.5ml each was pipetted into both the reagent blank (B) and

sample test (T) test tubes respectively. The serum sample of 0.1 ml was added to the sample test (T)

test tubes only and mixed thoroughly. Then, 0.1 ml of distilled water was added to the reagent blank

(B). Then, the entire reaction medium was well mixed and incubated at 37oC for 30 minutes in a water

bath.

A volume of 0.5 ml of 2, 4-dinitrophenylhydrazine (0.5 ml) was added to the reagent blank (B) and the

sample test tubes immediately after incubation. Also, 0.1 ml of the sample was added to blank (B) only.

The medium was mixed and allowed to stand for exactly 20 minutes at 250C. Finally, 5.0 ml of sodium

hydroxide (NaOH) solution was added to both the blank (B) and sample test (T) test tubes and mixed

thoroughly. The absorbance of sample Asample was read at a wavelength of 550nm against the sample

S/N Content Initial Concentration of Reagents

1. Phosphate Buffer 100 mmol/l, pH 7.4

L – Aspartate 100 mml/l

α – Oxoglutarate 2 mmol/l

2. 2, 4-Dinitrophenylhydrazine 2mmol/l

151

blank after 5 minutes. The activity of AST in mice serum was obtained from the already calibrated

table below (Randox Company).

2.2.7.10 Assay of Alanine Aminotransferase (ALT) Activity

The activity of alanine aminotransferase was assayed by the method of Reitman and Frankel (1957) as

outline in Randox kit.

Alanine aminotransferase assay, according to this method, is based on the principle that pyruvate is

formed from the reaction below:

α -Oxoglutarate + L-Alanine L-Glutamate + Pyruvate

Alanine aminotransferase activity was assayed by monitoring the concentration of pyruvate hydrazone

formed with 2, 4-dinitrophenylhydrazine.

Kit Reagents

P

roce

dur

es

The

ALT

substrate phosphate buffer of 0.5ml each was pipetted into two sets of test tubes labeled B (sample

blank) and T (sample test) respectively. The serum sample of 0.1 ml was added to the sample test (T)

test tubes only and mixed thoroughly and then, incubated exactly for 30 minutes in a water bath at

temperature of 370C.

A volume of 0.5 ml each of 2, 4-dinitrophenylhydrazine was added to both test tubes labeled T (sample

test) and B (sample blank) immediately after the incubation. Also, 0.1 ml of serum sample was added

to sample blank (B) only. The entire medium was mixed thoroughly and allowed to stand for exactly 20

minutes at 250C. After this, 5.0 ml each of sodium hydroxide (NaOH) solution was added to both test

tubes and mixed thoroughly. Absorbance of ASample against the sample blank was read at a wavelength

S/N Content Initial Concentration of Reagents

1.

Phosphate Buffer 100 mmol/l, pH 7.4

L – alanine 200 mmol/l,

Α – Oxoglutarate 2.0 mmol/l

2. 2, 4-Dinitrophenylhydrazine 2.0mmol/l

ALT

152

of 550nm against the sample blank after 5 minutes. The activity of ALT in the serum was obtained

from the already calibrated table below:

2.2.7.11 Assay of Alkaline Phosphatase (ALP) Activity

The activity of alkaline phosphatase (ALP) was assayed by the method of Klein et al. (1960) as outline

in Randox kit.

This method is based on the principle that serum alkaline phosphatase hydrolyses a colourless substrate

of phenolphthalein monophosphate giving rise to phosphoric acid and phenolphthalein, which at

alkaline pH value turns into a pink colour whose optical density can be measured

spectophotometrically.

Reagent Concentration

2-Amino-2-methyl-1-propanol pH 11 7.9 M

Phenolphthalein monophosphate 63mM

Na2HPO4 80 mM

Procedure

Distilled water (1.0 ml) was pipetted into 2 sets of test tubes labelled SA (sample) and ST (standard)

respectively. Then, one drop each of chromogenic substrate was added to the distilled water in the two

sets of test tubes. Their contents were mixed and incubated at 370C for 20 minutes in a water bath; after

which a standard solution of 0.1 ml was added to the standard test tube (ST) only, followed by the

addition of the serum sample of 0.1 ml to the sample test tube (SA). The content was also mixed and

incubated at 370C for 20 minutes in a water bath. A colour developer of 5.0 ml was added to both sets

of test tubes. The absorbance of the sample against the blank (water) was read at the wavelength of

550nm. The activity of alkaline phosphatase in the serum was obtained from the formula (calculations)

below:

30..

..

DOSA

DOSA = U/L of Alkaline phosphatase

Where

SA O.D = Sample Optical Density

ST O.D = Standard Optical Density

153

2.2.7.12 Determination of Total Cholesterol Concentration

Total cholesterol concentration was determined by the method of Allain et al. (1976) using Randox kit.

The total cholesterol using Randox Commercial kit is based on the principle that cholesterol is

determined after enzymatic hydrolysis and oxidation. The indicator quinoneimine is formed from

hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxide.

Cholesterol-ester + H2O cholesterol + Fatty acid (1)

Cholesterol-ester + H2O cholestene-3-one + H2O (2)

2H2O2 + Phenol + 4-Aminoantipyrene H2O Quinoneimine + H2O (3)

REAGENTS COMPOSITION

Content Reagents Initial Concentration of Solution

4-aminoantipyrine 0.3 mmol/l

Phenol 6 mmol/l

Peroxide ≥ 0.5 U/ml

Cholesterol esterase ≥ 0.15 U/ml

Cholesterol oxidase ≥ 0.1 U/ml

Pipes Buffer 80 mmol/l; pH 6.8

Standard 5.17 mmol/l (200 mg/dl)

Procedure

Distilled (10µl) water was pipetted into test tubes labeled B (reagent blank) only. Standard solution of

10µl was pipetted into test tubes labelled ST (standard). Serum sample (10µl) from the various

groups/mice was correspondingly pipetted into the last set of test tubes labelled SA (sample). Finally, 1

ml of the reagent was added to all the three sets of test tubes (Reagent blank, standard and sample),

mixed thoroughly and incubated for 10 minutes at 250C. The absorbance of sample (Asample) against the

reagent blank was read or measured at 500nm within 60 minutes.

154

The concentration of cholesterol in the serum sample was determined as follow; Conc. of

Cholesterol of in Sample = Standard

Sample

A

A× Conc. of Standard

2.2.7.13 Determination of High-Density Lipoproteins (HDL)–Cholesterol Concentration

High density lipoprotein (HDL) concentration was determined by the method of Albers et al. (1978)

using Randox kit.

The HDL-cholesterol determination using biosystem commercial kit method was based on the principle

that very low density lipoproteins (VLDL) and low density lipoprotein (LDL) in the sample

precipitated with phosphotungstate and magnesium ions. The supernatant contains high density

lipoproteins (HDL). The HDL cholesterol was spectrophotometrically measured.

The procedure took two steps;

a. Precipitation step: The sample (0.2 ml) was pipetted into labelled centrifuge tubes.

Also, 0.5 ml of reagent A (Phosphotungstate 0.4 mmol/l, magnesium chloride

20mmol/l) was added to the same sets of centrifuge tubes. The contents of the tubes

were thoroughly mixed and allowed to stand for 10 minutes at room temperature, then

centrifuged for 10 minutes at the minimum of 4000 rpm. The supernatant was carefully

collected.

b. Colorimetric step:Reagent B was brought to room temperature. Distilled water (50µl)

was pipetted into the blank test tubes (B). HDL cholesterol standard (50µl) and sample

supernatant were pipetted into the standard (ST) and the sample (SA) test tubes

respectively. Reagent B (1.0µl) each was added to all test tubes and thoroughly mixed;

then incubated for 30 minutes at room temperature. The absorbance (A) of the standard

and sample at 500nm wavelength was measured against the blank. The colour was stable

for at least 30 minutes.

Calculations

The HDL cholesterol concentration in the sample was calculated using the following general formula:

Standard

Sample

A

A × 52.5 = mg/dl HDL-cholesterol

Reagent Contents and Composition

155

Reagent A: 2 x 50ml phosphotungstate 0.4 mmol/l, magnesium chloride 20 mmol/l

Reagent B: 2 x 50 ml phosphate 35 mmol/l, cholesterol esterase > 0.2 U/ml, cholesterol oxidase > 0.1

U/ml, peroxidase > 1 U/ml, 4-aminoantipyrine 0.5 mmol/L, sodium cholate 0.5 mmol/l,

dichlorophenol-sulfonate 4 mmol/L, pH 7.0. HDL cholesterol standard: 1 x 5 ml. cholesterol 15 mg/dl;

aqueous primary standard.

2.2.7.14 Determination of Triacylglycerol Concentration

Triacylglycerol (TAG) concentration was determined by the method of Allain et al. (1976) using

Randox kit.

Principle

The triacylglycerol concentration was determined after enzymatic hydrolysis with lipases. The

indicator is a quinoneimine formed from hydrogen –peroxide, 4-aminophenazone and 4-chlorophenol

under the catalytic influence of peroxide.

Triglycerides + H2O Lipases

Glycerol + fatty acids

Glycerol + ATP GK

Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 GPO

Dihydroxyacetone + phosphate + H2O2

2H2O2 + 4-aminophenazone + 4-chlorophenol POD

Quinoneimine + HCl + 4H2O.

A known volume of 100µl of the reagent was pipetted into the reagent blank tube, standard tube and

the sample tubes. 10µl of the standard was then added to the standard tube while 10µl of the sample

was pipetted into the sample tube. The mixtures in the three tubes were mixed and incubated at 20 –

250C for 10 minutes. Then, the absorbance of the sample and the standard were measured against the

reagent blank within 60 minutes at 546nm.

Triacylglycerol concentration (mmol/l) = Standard

Sample

A

A × 2.29

ASample = Absorbance of sample

AStandard= Absorbance of standard

2.2.7.15 Determination of Low Density Lipoprotein-Cholesterol Concentration

156

Low density lipoprotein (LDL) concentration was determined by the method of Assmann et al.

(1984) using Randox kit.

The LDL-cholesterol determination, using Randox Commercial Kit is based on the principle that low

density lipoproteins (LDL) are precipitated by heparin or EDTA at their isoelectric point (pH 5.04).

After centrifugation, the high density lipoproteins (HDL) and the very low density lipoproteins (VLDL)

remain in the supernatant. These were determined by enzymatic methods.

LDL-cholesterol = Total cholesterol – cholesterol in the supernatant

Reagents

CONTENT INITIAL CONCENTRATION OF SOLUTION

Precipitation reagent Heparin 50,000 IU/L

Sodium 0.064 mol/L, pH 5.04

Procedure

The serum (100µl) was pipetted into the centrifuge tube which was immediately accompanied with the

addition of 1 ml of the precipitation reagent to the centrifuge tube. The contents were well mixed and

left to stand for 10 minutes at 25oC; then, centrifuged for 15 minutes at 3500 rpm. The cholesterol

concentration of the supernatant was determined within 1 hour after centrifugation.

Distilled water (50µl) was pipetted into a reagent blank test tube (B) of a new set of test tubes. The

standard solution (50µl) each and the supernatant were pipetted into the standard test tube (ST) and the

sample test tube (SA) respectively. A volume of 1ml each of the reagent solution was added to all the

three sets of test tubes. The test tubes contents were mixed and inoculated for 10 minutes at 25oC. The

absorbance of the sample (Asample) against the reagent blank was read at;

Conc. of Cholesterol in the Supernatant Standard

Sample

A

A × Conc. of Standard

Calculation of the LDL-cholesterol:

LDL-Cholesterol = Total Cholesterol – Cholesterol in the Supernatant

2.2.7.16 Acute Toxicity Studies (LD50)

157

Acute toxicity studies (LD50) was measured using method of Lorke (1989). The animals were

divided into two groups, A and B, with each group subdivided into four groups made up of three

animals each.

Experimental Protocol for Acute Toxicity Studies

Phase I:

Group 1 : Mice were administered with 10mg/kg of body weight of the ethanol leaf extract of

Moringa oleifera.

Group 2 : Mice were administered with 100mg/kg of body weight of the ethanol

leaf extract of Moringa oleifera.

Group 3 : Mice were administered with 1000 mg/kg of body weight of the ethanol leaf extract of

Moringa oleifera.

Group 4 : Mice were administered with 1000 mg/kg of body weight of distilled water.

Phase II

Group 1 : Mice were administered with 1900 mg/kg of body weight of the ethanol leaf extract.

Group 2 : Mice were administered with 2600 mg/kg of body weight of the ethanol leaf extract.

Group 3 : Mice were administered with 5000 mg/kg of body weight of the ethanol leaf extract.

Group 4 : Mice were administered with 5000 mg/kg of body weight of distilled water.

The mice were monitored closely for 24 hours for signs of toxicity and lethality

2.2.8 Phytochemical Analyses

Phytochemical analyses were carried out according to the methods of Harborne (1973) and Trease and

Evans (1989).

The following phytochemical tests were carried out:

2.2.8.1 Test for Carbohydrate (Molisch’s Test)

A known weight, 0.1 g, of extract was boiled with 2ml of water and filtered. To the filtrate, few drops

of naphtol solution in ethanol (Molisch’s reagent) were added. Concentrated sulphuric acid was then

gently poured down the side of the test tube to form a lower layer

158

2.2.8.2 Test for Alkaloids (General Tests)

Sulphuric acid (20 ml of 5%) in 50% ethanol was added to about 2g of the powdered material (extract)

and heated on a boiling water bath for 10 minutes, cooled and filtered. The filtrate (2 ml) was tested

with a few drops of:

Mayer’s reagent (potassium mercuric iodide solution)

Dragendorff’s reagent (bismuth potassium iodide solution)

Wagner’s reagent (iodide in potassium iodide solution)

Picric acid solution (1%)

The remaining filtrate was placed in 100ml separating funnel and alkaline with diluted ammonia

solution. The aqueous alkaline solution was separated and extracted with two 5 ml portions of diluted

sulphuric acid. The extract was tested with a few drops of Mayer’s Wagner’s and Drangendorff’s

reagent.

2.2.8.3 Test for Glycosides (Fehling’s Test)

A known volume, 5 ml, of a mixture of equal parts of Fehling’s solution I and II were added to 5 ml of

the aqueous extract and then heated on a water bath for 5 minutes.

2.2.8.4 Test for Saponins (Fehling’s Method)

Distilled water (20 ml) was added to 0.25g of extract in 100ml beaker and boiled gently on a hot water

bath for 2 minutes. The mixture was filtered hot and allowed to cool and the filtrate used as follows:

To 5ml of the filtrate was added 5ml of Fehling’s solution (equal parts of I and II) and the content

heated. A reddish precipitate indicated the presence of saponins. It was then heated further with

sulphuric acid.

2.2.8.5 Test for Tannins (Ferric Chloride Method)

A known weight of 1g of the powdered material (extract) was boiled with 50ml of water, filtered and

used for the ferric Chloride Test proper:

To 3 ml of the filtrate, few drops of ferric chloride were added.

159

2.2.8.6 Test for Flavonoids (Ammonium Test Method)

Ethylacetate (10ml) were added to 0.2g of the plant extract and heated on a water bath for 3 minutes.

The mixture was cooled, filtered and the filtrate used for the ammonium test proper:

A volume of 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. The layers were

allowed to separate.

2.2.8.7 Test for Resins (Precipitaion Test)

The Moringa oleifera leaf extract (0.2g) was extracted with 15 ml of 95% ethanol. The alcoholic

extract was then poured into 20ml of distilled water in a beaker.

2.2.8.8 Test for Proteins (Million’s Test)

Two drops of Million’s reagent were added to the filtrate in a test tube.

2.2.8.9 Test for Oils

The Moringa oleifera leaf extract (0.1g) material was pressed between a filter paper and the filter paper

was put under serious observation.

2.2.8.10 Test for Steroids and Terpenoids

Ethanol (9ml) was added to 1g of the plant extract and refluxed for few minutes and filtered. The

filtrate was concentrated to 2.5 ml on a boiling water bath and 5 ml of hot water was added. The

mixture was allowed to stand for 1 hour and the waxy matter filtered off. The filtrate was extracted

with 2.5 ml of chloroform using separating funnel. To 0.5 ml of the chloroform extract in a test tube

was carefully added 1 ml of concentrated sulphuric acid to form a lower layer. Another 0.5 ml of the

chloroform extract was evaporated to dryness on a water bath and heated with 3 ml of concentrated

sulphuric acid for 10 minutes on a water bath.

2.3 STATISTICAL ANALYSIS

The data obtained from the laboratory tests were subjected to one- way analyses of variance (ANOVA).

Significant differences were obtained at p≤0.05.The results were expressed as mean and standard

160

deviation (SD).This analysis was estimated using computer software known as Statistical Package

for Social Sciences (SPSS), version 18.

161

CHAPTER THREE

RESULTS

3.1 Phytochemical Constituents of Moringa oleifera

Phytochemical analyses of ethanol extract of Moringa oleifera leaf extract showed the presence

of tannins, carbohydrates, saponins, glycosides, reducing sugars, terpenoids, steroids, flavonoids and

alkaloids. Phytochemicals such as resins, proteins, and fats and oil were not detected during the test.

The results presented in the Table 4 below show that flavonoids were more in quantity than other

phytochemicals detected. Phytochemicals such as carbohydrates, reducing sugars, steroids and

alkaloids were moderate in concentration while phytochemicals such as tannins, saponins, glycosides

and terpenoids were found to be relatively low in concentration.

Table 4: Phytochemical constituents of Moringa oleifera

CONSTITUENTS ETHANOL EXTRACT

Tannins +

Carbohydrates ++

Saponins +

Glycosides +

Reducing Sugars ++

Terpenoids +

Steroids ++

Flavonoids +++

Alkaloids ++

Resins ND

Proteins ND

Fats and Oil ND

+++ = Relative Abundance of Compound

++ = Moderate Abundance of Compound

+ = Relative low Presence of Compound

ND = Not Detected.

162

3.2 Acute Toxicity (LD50)

The LD50 of the ethanol extract in mice was found to be more than 2600mg/kg

and less than 5000 mg/kg body weight. One animal died and the other remaining two

animals in the group showed signs of toxicity as illustrated below within 24 hours of

constant observation.

PHASE 1

Group Dosage Mice 1 Mice 2 Mice 3

Group 1 10 mg/kg ND and NST ND and NST ND and NST

Group 2 100 mg/kg ND and NST ND and NST ND and NST

Group 3 1000 mg/kg ND and NST ND and NST ND and NST

Group 4 Standard Control ND and NST ND and NST ND and NST

PHASE 2

Group Dosage Mice 1 Mice 2 Mice 3

Group 1 1,900 mg/kg ND and NST ND and NST ND and NST

Group 2 2,600 mg/kg ND and NST ND and NST ND and NST

Group 3 5,000 mg/kg ST D ST

Group 4 Standard Control ND and NST ND and NST ND and NST

ND = No Death

NST = No Signs of Toxicity

163

D = Death

ST = Signs of Toxicity

3.3. Effect of Ethanol Leaf Extract of Moringa oleifera on Percentage Parasitaemia

Fig. 2 shows that 3 days of inoculation the mean values for percentage parasitaemia of mice in

groups 4 and 5 significantly decreased (p<0.05) compared to the values of mice in groups 1 (positive

control), 2 and 3. On day 3 of treatment the mean values for percentage parasitaemia in all the groups

significantly decreased (p<0.05) compared to the mean percentage parasitaemia of mice in groups 1

(positive control) and 2. Also, on day 5 of treatment the mean percentage parasitaemia in all the groups

significantly decreased (p<0.05) compared to the values for the percentage parasitaemia of mice in

group 1 (positive control). On day 28 of post treatment the mean values of the percentage parasitaemia

significantly decreased in all the groups compared to the mean percentage parasitaemia of group 1

(positive control). Finally on day 28 of post treatment also showed significant (p<0.05) clearance of the

parasitaemia in groups 4 and 5 compared to the mean values of the percentage parasitaemia in groups 1

(positive control), 2 and 3 animals.

164

Fig. 2: Effect of ethanol leaf extract Moringa oleifera on percentage

parasitaemia in mice

0

1

2

3

4

5

6

7

8

9

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

Pa

ras

ita

em

ia (

%)

3 Days of Inoculation

Day 3 of Treatment

Day 5 Treatment

Day 28 of Post-Treatment

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

165

3.4 Effect of Ethanol Leaf Extract of Moringa oleifera on Haemoglobin Concentration in

Mice)

Fig. 3 shows that 3 days after inoculation the mean values for haemoglobin in all the groups

were essentially similar, while the value obtained for group 4 was significantly (p<0.05) lower than for

mice in group 1 (positive control) . On day 5 of treatment the mean values for haemoglobin in groups

4, 5 and 6 significantly increased (p<0.05) compared to group1 (positive control). Finally, on day 28 of

post treatment the mean values for heamoglobin in groups 4, 5 and 6 (negative control) significantly

increased (p<0.05) compared to group 1 (positive control).

166

Fig. 3: Effect of ethanol leaf extract of Moringa Oleifera on haemoglobin concentration in mice

0

2

4

6

8

10

12

14

16

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Day 3 After Inoculation Day 5 of Treatment Day 28 of Post-Treatment

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

Me

an

Hb

Co

nc

.

(g/d

l)

167

3.5 Effect of Ethanol Leaf Extract of Moringa oleifera on Total White Blood Cell Count in

Mice

Fig. 4 shows the effect of ethanol leaf extract of Moringa oleifera on total white blood cell

count. The TWBC (baseline) count obtained 3 days after inoculation for mice in groups 1, 2, 3, 5 and 6

were essentially similar, while the value obtained for mice in group 4 was significantly lower than that

for mice in group 1. On day 5 after commencement of treatment, mean value for group 2 mice was

significantly (p<0.05) lower than that of group 1 mice, while mean values for mice in groups 3 and 5

were significantly (p<0.05) higher than that of group 1 mice. There was no significant difference

(p>0.05) between the mean values for mice in groups 4 and 6 when compared with that for mice in

group 1. TWBC count on day 28 of treatment in group 3 mice was essentially similar to the value of

TWBC in group 6 (negative control) mice, while the values obtained for mice in groups 2, 3, 4, 5 and 6

were significantly (p<0.05) higher than that for group 1 (positive control) mice.

168

Fig. 4: Effect of ethanol leaf extract of Moringa oleifera on total

white blood cell count in mice

0

5

10

15

20

25

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

To

tal

WB

C (

10

9/L

)

Day 3 After Inoculation

Day 5 of Treatment

Day 28 of Post-Treatment

3.6 Effect of Ethanol Leaf Extract of Moringa oleifera on Packed Cell Volume in Mice

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

169

Fig. 5 shows that 3 days after inoculation mean values for PCV of mice in groups 2, 3, 5,

and 6 were not significantly (p>0.05) different from the value obtained for mice in group 1 (positive

control); but the value obtained for mice in group 4 was significantly (p<0.05) lower than that for mice

in group 1 (positive control).On day 5 of treatment , mean values for PCV for groups 2 and 3 were

essentially similar to that of animals in group 1. On the other hand, values obtained for mice in groups

4, 5 and 6 showed significant (p<0.05) increases above the value for animals in the group 1 (positive

control). For day 28 post treatment, whereas the mean PCV values for groups 4, 5 and 6 animals were

significantly (p<0.05) higher than that of group 1 mice, mean values for those in groups 2 and 3

showed no significant (p>0.05) difference when compared with the value for group 1 mice. Also, mean

PCV values for mice in groups 4, 5 and 6 were similar.

170

Fig. 5: Effect of ethanol leaf extract of Moringa oleifera on packed

cell volume in mice

0

5

10

15

20

25

30

35

40

45

50

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

PC

V (

%)

Day 3 After Inoculation

Day 5 of Treatment

Day 28 of Post-Treatment

3.7 Effect of Ethanol Leaf Extract of Moringa oleifera on Red Blood Cell Count in Mice

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

171

Fig. 6 shows that mean RBC baseline obtained 3 days after inoculation for mice in groups

2, 3, 4, 5 and 6 (negative control) mice were not significantly (p>0.05) different compared to the value

in group 1 (positive control) mice. On day 5 of treatment showed significant increase (p<0.05) in RBC

count of groups 2, 3, 4, 5 and 6 (negative control) mice compared to the mean value for RBC count of

mice in group 1 (positive control). Also day 28 of post treatment, showed significant increase (p<0.05)

in the mean values of RBC count of mice in groups 2, 3, 4, 5 and 6 (negative control) mice when

compared to the value of RBC count obtained for group 1 (positive control) mice. But the mean value

of RBC obtained in group 4 mice was essentially similar to that for group 6 (negative control).

172

Fig. 6: Effect of ethanol leaf extract of Moringa oleifera on red blood

cell count in mice

0

2

4

6

8

10

12

14

16

18

20

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

RB

C C

ou

nt

(x1

06)

Day 3 After Inoculation

Day 5 of Treatment

Day 28 of Post-Treatment

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

173

3.8 Effect of Ethanol Leaf Extract of Moringa oleifera on Serum Creatinine

Concentration in Mice

Fig. 7 shows that on day 28 of post treatment mean serum creatinine concentration of mice in

groups 2, 3, 4, 5 and 6 (negative control) were significantly (p<0.05) lower than that of the group 1

(positive control). Also, the mean serum creatinine concentrations of groups 5 and 6 mice were similar

when compared.

174

Fig. 7: Effect of ethanol leaf extract of Moringa oleifera on creatinine

concentration in mice

0

10

20

30

40

50

60

70

80

90

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

Cre

ati

nin

e C

on

c (

μm

ol/

L)

3.9 Effect of Ethanol Leaf Extract of Moringa oleifera on Urea Concentration in Mice

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

175

Fig. 8 shows that on day 28 of post treatment mean values for urea concentration of mice in

groups 2, 3, 4, 5 and 6 (negative control) were not significantly (p>0.05) different from the value

obtained for mice in group 1 (positive control). But the values obtained for mice in groups 2 and 4 were

significantly (p<0.05) lower than that for mice in group 6 (negative control). Also mean values for urea

concentration in groups 1 (positive control) and 3 were similar when compared.

176

Fig. 8: Effect of ethanol leaf extract of Moringa oleifera on urea

concentration in mice

0

1

2

3

4

5

6

7

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

Ure

a C

on

c (

mm

ol/

L)

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

177

3.10 Effect of Ethanol Leaf Extract of Moringa oleifera on Total Bilirubin Concentration in

Mice

Fig. 9 shows that on day 28 of post treatment mean values for total bilirubin

concentration of mice in groups 3, 4, 5 and 6 significantly decreased (p<0.05) in a dose-

dependent pattern when compared to the mean values of total bilirubin concentration

of mice in groups 1 (positive control) and 2. The mean values obtained for total

bilirubin concentrations for mice in group 5 and 6 (negative control) were similar when

compared.

178

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

179

3.11 Effect of Ethanol Leaf Extract of Moringa oleifera on Alanine

aminotransferase Activity in Mice

Fig. 10 shows that on day 28 of post treatment mean values for ALT activity for

mice in groups 3, 4, 5 and 6 (negative control) were significantly (p<0.05) lower than

that for groups 1 and 2. Meanwhile, the mean value for ALT activity of mice in group 2

was significantly (p<0.05) lower than that for group 1.But the mean ALT values for

groups 3, 4 and 5 were essentially similar.

180

Fig. 10: Effect of ethanol leaf extract of Moringa oleifera on Alanine

aminotransferase activity in mice

0

5

10

15

20

25

30

35

40

45

50

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

AL

T A

cti

vit

y (

IU/L

)

3.12 Effects of Ethanol Leaf Extract of Moninga oleifera on Aspartate aminotransferase Activity in Mice

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

181

Fig. 11 shows that on day 28 of post treatment mean values for AST activity

in mice for groups 2, 3, 4 and 5 were similar with the values for mice in groups 1

(positive control) and 6 (negative control).

182

183

Fig. 11: Effect of ethanol leaf extract of Moringa oleifera on

Aspartate aminotransferase activity in mice

0

50

100

150

200

250

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

AS

T A

cti

vit

y (

IU/L

)

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

184

3.13 Effect of Ethanol Leaf Extract of Moringa oleifera on Alkaline Phosphatase Activity in Mice

Fig. 12 shows the effect of ethanol leaf extract of Moringa oleifera on alkaline phosphatase activity in mice. On day 28 of post

treatment shows that the mean ALP values for mice in groups 2, 3, 5 and 6 (negative control) were significantly (p<0.05) lower than

that for mice in groups 1 (positive control) and 4. But the ALP values for mice in groups 4 and 1 (positive control) were similar when

compared.

185

Fig. 12: Effect of ethanol leaf extract of Moringa oleifera on Alkaline

phosphatase activity in mice

0

20

40

60

80

100

120

140

160

180

200

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

AL

P A

cti

vit

y (

IU/L

)

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

186

3.14 Effect of Ethanol Leaf Extract of Moringa oleifera on Total Cholesterol

Concentration in Mice

Fig. 13 shows that on day 28 of post treatment mean total cholesterol

concentration for mice in groups 2, 3, 4 and 5 were non-significantly (p>0.05) lower

than the values for mice in groups 1 (positive control) and 6 (negative control).

187

188

Fig. 13: Effect of ethanol leaf extract of Moringa oleifera on total

cholesterol concentration in mice

0

0.5

1

1.5

2

2.5

3

3.5

4

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

Ch

ol

Co

nc

. (m

mo

l/L

)

3.15 Effect of Ethanol Leaf Extract of Moringa oleifera on Total High Density Lipoprotein Concentration in Mice

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

189

Fig. 14 shows that on day 28 of post-treatment, there was a non-significant

(p>0.05) increase in the high density lipoprotein (HDL) concentration of the mice in all

the test groups administered graded doses of the extract (45, 90 and 180 mg/kg body

weight) when compared to the HDL concentration of mice in the three control groups

(positive, negative and standard). In the same vein, the HDL concentration of mice in

groups 5 (standard control) and 6 (negative control) decreased compared to the HDL

concentration of mice in group 1 (positive control). However, such decrease was found

to be non-significant (p>0.05) as shown in Fig. 14.

190

191

Fig. 14: Effect of ethanol leaf extract of Moringa oleifera on total

high density lipoprotein concentration in mice

0

0.2

0.4

0.6

0.8

1

1.2

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

HD

L C

on

c.

(mm

ol/

L)

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

192

3.16 Effect of Ethanol Leaf Extract of Moringa oleifera on Low Density

Lipoprotein Concentration in Mice

Fig.15 shows that on day 28 of post treatment mean values of LDL concentration

for mice in groups 2 , 3 , 4 and 5 were non-significantly (p>0.05) lower than that for

mice in groups 1 (positive control) and 6 (negative control). But the mean value

obtained for LDL of mice in group 6 was similar to that for group 1 (positive control)

when compared.

193

Fig. 15: Effect of ethanol leaf extract of Moringa oleifera on low

density lipoprotein concentration in mice

0

0.5

1

1.5

2

2.5

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

LD

L C

on

c.

(mm

ol/

L)

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

194

3.17 Effect of Ethanol Leaf Extract of Moringa oleifera on Triacylglycerol

Concentration in Mice

Fig. 16 shows that the mean TAG concentrations of mice in groups 2, 3, and 5

were non-significantly (p>0.05) lower than the values for mice in groups 1 (positive

control), 4 and 6 (negative control). But the mean TAG value for mice in group 1

(positive control) was similar to that in group 6 (negative control) when compared.

195

196

Fig. 16: Effect of ethanol leaf extract of Moringa oleifera on

triacylglycerol concentration in mice

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Group

Me

an

TA

G C

on

c.

(mm

ol/

L)

Group 1=Positive Control Group 4=180mg/kg b.w. of Moringa oleifera

Group 2=45mg/kg b.w. of Moringa oleifera Group 5=5mg/kg b.w. of Artesunate

Group 3=90mg/kg b.w. of Moringa oleifera Group 6=Negative Control

197

CHAPTER FOUR

DISCUSSION

Malaria is a major public health problem and cause of much suffering and premature death in the

poorer areas of the Tropical Africa, Asia and Latin America. Human beings are exposed to malaria

through the bite of an infected female anopheles mosquito, blood transfusion and congenitally from

mother to her child (Bruce, 1981). In many endemic areas, it is becoming difficult to control, because

of the parasite resistance to antimalarial drugs and the failure of vector control measures. Due to

resistance to some of the conventional drugs used for the treatment of malaria and the impact of

malaria to world health, it is therefore necessary to search for new, cheap and easily available drug that

will be used for the treatment of malaria (Dondorp, 2007). The medicinal uses of many plants like

Moringa oleifera cannot be over-emphasised. The choice of this plant for the research work was based

on its numerous ethnomedicinal properties. The results from the phytochemical studies of the ethanol

leaf extract of Moringa oleifera indicated the presence of flavonoids, steroids, tannins, carbohydrates,

saponins, glycosides, alkaloids and phenols which may play a role in the metabolism of the plant. The

LD50 of the extract was found to be less than 5000 mg/kg but more than 2,600mg/kg body weight of the

extract.

The observation on the effect of ethanol leaf extract of Moringa oleifera on percentage parasitaemia in

mice showing a significant (p<0.05) clearance of parasitaemia in group 4 (180mg/kg body weight of

the extract) and group 5 (5mg/kg body weight of artesunate) when compared to group 1 (positive

control) is consistent with the findings of Monzon (1995) in Phillipines, who administered Moringa

oleifera leaf extract in mice that were infected with malaria and other parasitic diseases.The result

showed that the extract might be effective against the parasites. The result also showed a significant

increase (p<0.05) in parasitaemia in group 1 (positive control) treated with 5mg/kg distilled water

which could lead to the destruction of the liver, blood cells, kidney and other vital organs in the mice

(Trampuz et al., 2003). This could be as a result of the infection of the liver by the sporozoites and the

resultant multiplication of the merozoites in the blood cells.

The result of the effect of ethanol leaf extract of Moringa oleifera on the haematological parameter of

packed cell volume showed a non significant difference in packed cell volume (p>0.05) in group 4 (180

mg/kg body weight of the extract) and group 5 (5 mg/kg body weight of the artesunate) compared to

198

group 6 (negative control). But, a significant reduction (p<0.05) in packed cell volume was

observed in group 1 (positive control) when compared to group 6 (negative control). This showed that

Moringa oleifera ethanol leaf extract has ameliorated the effect of malaria parasitaemia on the packed

cell volume. This agrees with the work of Ambi et al., (2006) who showed that Moringa oleifera leaf

extract boosted heamatological parameters of packed cell volume in rats. Packed cell volume is used to

asses anaemia, erythrocytosis, haemodilution and haemoconcentration.A decrease in packed cell

volume indicates anaemia (Dacie and Lewis, 2000).

The result of the effect of ethanol leaf extract of Moringa oleifera on red blood cell count showed a non

significant difference (p>0.05) in group 4 (180 mg/kg body weight of the extract) compared to group 6

(negative control).This, also corroborates with the work of Ambi et al. ,(2006) showing that Moringa

oleifera leaf extract boost red blood cell counts in rats.. There was a significant increase (p<0.05) in red

blood cell count in group 4 (180mg/kg body weight of the extract) when compared to group 1 (positive

control) . A decrease in red blood cell could be as a result of anaemia (Dacie and Lewis, 2000).

Moringa oleifera ethanol leaf extract has probably repaired the damages caused by merozoites to the

red blood cell in mice that were infected with malaria.

The effect of ethanol leaf extract of Moringa oleifera on haemoglobin concentration in mice showed a

significant increase (p<0.05) in haemoglobin in group 4 (180 mg/kg body weight of the extract), group

5 (5mg/kg body weight of the artesunate) and group 6 (negative control) when compared to group 1

(positive control). But, group 4 (180mg/kg body weight of the extract) and group 5 (5 mg/kg body

weight of the artesunate) showed no significant difference (p>0.05) in haemoglobin concentration

when compared to group 6 (negative control). This corroborated with the work of Ambi et al. (2006)

who showed that Moringa oleifera leaf extract boosted haemoglobin concentration in rat. A complete

blood count is used to asses symptoms such as weakness, fatigue, anaemia, infection and other

disorders. Haemoglobin molecule fills up the red blood cells. It transports oxygen and gives the blood

cell its red colour. The higher the haemoglobin concentration, the higher its ability to transport oxygen

throughout the body.

The effect of ethanol leaf extract of Moringa oleifera on total white blood cell count showed a

significant increase (p<0.05) in total white blood cell count in other groups when compared to group 1

(positive control) .The Moringa oleifera ethanol leaf extract increased the total white blood cell in

group 2 (45 mg/kg body weight of the extract), group 3 (90 mg/kg body weight of the extract) and

199

group 4 (180 mg/kg body weight of the extract) when compared to group 1 (positive control). This

is also consistent with the work of Ambi et al., (2006) that showed the potency of Moringa oleifera leaf

extract in increasing white blood cell counts in rat. This could be the reason for reduced parasitaemia in

groups 2 (45mg/kg body weight of the extract) and group 3 (90 mg/kg body weight of the extract) and

total clearance of the parasitaemia in group 4 (180 mg/kg body weight of the extract) and group 5 (5

mg/kg body weight of the artesunate).

The effect of ethanol leaf extract of Moringa oleifera on serum creatinine concentration in mice

showed a significant decrease (p<0.05) in serum creatinine concentration in groups ( 2,3,4,5 and 6)

treated with 45,90,180 mg/kg body weight of the extract ,5mg/kg body weight of artesunate and

5mg/kg body weight of distilled water respectively were compared to group 1 (positive control).This

showed that the ethanol leaf extract of Moringa oleifera has reduced the level of serum creatinine in

group 2 (45 mg/kg body weight of the extract) ,group 3 (90 mg/kg body weight of the extract) and

group 4 (180 mg/kg body weight of the extract) thereby ameliorating the effects of malaria

parasitaemia on the kidney. There was no significant difference (p>0.05) in serum creatinine when

group 4 (180 mg/kg body weight of the extract) was compared to group 3 (90 mg/kg body weight of

the extract) and group 2 (45 mg/kg body weight of the extract). This is in line with the findings of

Mazumder et al. (1999) who showed the hepatorenal function of Moringa oleifera on mice.

The effect of ethanol leaf extract of Moringa oleifera on urea concentration in mice showed a non

significant difference (p>0.05) in urea concentration in groups (2, 3, 4, 5 and 6) treated with with 45,

90, 180 mg/kg body weight of the extract, 5mg/kg body weight of artesunate and 5mg/kg body weight

of distilled water respectively were compared to group 1 (positive control). This showed that malaria

had no effect on urea concentration. But there was a significant decrease (p<0.05) in urea concentration

when group 2 (45mg/kg body weight of the extract) and group 4 (180 mg/kg body weight of the

extract) were compared to group 6 (negative control). This corroborates with the work of Mazumder et

al. (1999) showing the potential effects of the ethanol leaf extract of Moringa oleifera on ameliorating

renal dysfunctions.

The effect of ethanol leaf extract of Moringa oleifera on alanine aminotransferase activity in mice

showed a significant decrease (p<0.05) in alanine aminotransferase in groups (3,4,5 and 6 ) treated

with 90, 180 mg/kg body weight of the extract ,5mg/kg body weight of artesunate and 5mg/kg body

weight of distilled water respectively were compared to the alanine aminotransferase of group 1

200

(positive control) and group 2 (45 mg/kg body weight of the extract). Alanine aminotransferase in

conjuction with aspartate aminotransferase is usually used to diagnose hepatocellular injury and

diseases. Alanine aminotransferase is cytosolic and is present in large concentrations in liver and, in

less amount in kidney, heart and skeletal muscle (Johnston, 1999). It is therefore a more specific liver

marker than aspartate aminotransferase (Song et al., 2004). This confirms the damage that was done to

the liver as a result of the malaria infection. But group 3 (90 mg/kg body weight of the extract), group 4

(180 mg/kg body weight of the extract) and group 5 (5 mg/kg body weight of the artesunate) all

showed no significant difference (p>0.05) in alanine aminotransferase compared to group 6 (negative

control). This showed the ameliorative effect of the liver damage by the Moringa oleifera ethanol leaf

extract as a result of the malaria parasitaemia. This agrees with the findings of Fakurazi et al. (2008)

and Alaaeldin (2009) that showed the preventive and ameliorative effects of Moringa oleifera on liver

injury and damages.

The effect of ethanol leaf extract of Moringa oleifera on aspartate aminotransferase activity in mice

showed no significant difference (p>0.05) in aspartate aminotransferase when group 6 (negative

control) was compared to other groups including group 1(positive control).This showed that malaria

parasitaemia, the extract and artesunate did not affect the aspartate aminotransferase activity in the

mice.

The effect of ethanol leaf extract of Moringa oleifera on alkaline phosphatase activity in mice showed a

significant decrease (p<0.05) in alkaline phosphatase in all the groups compared to group 1 (positive

control) and group 4 (180 mg/kg body weight of the extract). Alkaline phosphatase is present in all the

tissues throughout the body, but is particularly concentrated in the liver, bile duct, kidney, bone and the

placenta. It is therefore not a specific liver marker. This result showed that malaria parasitaemia had

effect on the alkaline phosphatase activity in the mice. But, in group 4 (180 mg/kg body weight of the

extract) the elevated level of alkaline phosphatase could be as a result of active bone formation

occurring as alkaline phosphatase is a by-product of osteoblast activity. But, group 2 (45 mg/kg body

weight of the extract) and group 3 (90 mg/kg body weight of the extract) showed a significant

reduction (p<0.05) in alkaline phosphatase which corroborated with the findings of Alaaeldin (2009)

and Fakurazi et al. (2008) who showed ameliorative effects of Moringa oleifera leaf extract on liver

injury.

201

The effect of ethanol leaf extract of Moringa oleifera on total bilirubin in mice showed a

significant decrease (p<0.05) in total bilirubin concentration of all the groups compared to the total

bilirubin concentration of group 1 (positive control) and group 2 (45 mg/kg body weight of the extract)

. This could be as a result of liver damage by the malaria parasitaemia. But in group 3 (90 mg/kg body

weight of the extract) and group 4 (180 mg/kg body weight of the extract) both showed a significant

reduction (p<0.05) in total bilirubin thereby ameliorating the effect of malaria on the liver. This agrees

with the findings of Alaaeldin (2009) and Pari and Kumar (2002) which showed the protective effects

of Moringa oleifera extract on the liver.

The effect of ethanol leaf extract of Moringa oleifera on total cholesterol concentration in mice

showed a non significant difference (p>0.05) in all the groups compared to group 6 (negative control).

This indicated that malaria had no effect on total cholesterol concentration of the mice. But, there was

non-significant decrease (p>0.05) in total cholesterol of group 2 (45 mg/kg body weight of the extract),

group 3 (90 mg/kg body weight of the extract), group 4 (180 mg/kg body weight of the extract) and

group 5 (5 mg/kg body weight of the artesunate) animals compared to group 6 (negative control)

animals. This result agrees with the works of Ghasi et al. (2000) and Mehta et al. (2003). Cholesterol is

essential for all animals’ life, high levels in blood circulation, depending on how it is transported within

lipoprotein, are strongly associated with progression of artherosclerosis and other cardiovascular

diseases.

The effect of ethanol leaf extract of Moringa oleifera on high density lipoprotein concentration in mice

showed non-significant difference (p>0.05) in high density lipoprotein concentration of all the groups

mice compared to the high density lipoprotein concentration of mice in group 6 (negative control). But,

there was a non-significant increase (p>0.05) in high density lipoprotein concentrations of group 2

(45mg/kg body weight of the extract), group 3 (90mg/kg body weight of the extract) and group 4

(180mg/kg body weight of the extract) mice. These are consistent with the work of Ghasi et al. (2000)

and Mehta et al. (2003). High density lipoprotein particles transport cholesterol back to the liver for

excretion or to other tissues that use cholesterol to synthesize hormones. So, having high concentrations

of high density lipoprotein correlated with better health outcomes.

The effect of ethanol leaf extract of Moringa oleifera on low density lipoprotein concentration in mice

showed a non-significant difference (p>0.05) in low density lipoprotein concentration of mice in all

the groups compared to the low density lipoprotein concentration of mice in group 6 (negative

202

control).However, there was a non-significant decrease (p>0.05) in low density lipoprotein (LDL)

concentration of groups 2 (45 mg/kg body weight of the extract), group 3 (90mg/kg body weight of the

extract), group 4 (180mg/kg body weight of the extract) and group 5 (5mg/kg body weight of the

artesunate) mice compared to group 6 (negative control). These findings are in line with the work of

Ghasi et al. (2000) and Mehta et al. (2003). High concentration of low density lipoprotein

(hypercholesterolemia) and lower concentration of functional high density lipoprotein are strongly

associated with cardiovascular diseases because these promote antheroma development in arteries

(antherosclerosis). This disease process leads to myocardial infarction (heart attack), stroke and

peripheral vascular diseases. Since higher low density lipoprotein particle concentrations and smaller

low density lipoprotein particle size contribute to this process more than the cholesterol content of the

low density lipoprotein particles, low density lipoprotein particles are called bad cholesterol because

they have been linked to antheroma formation. On the other hand, high concentrations of functional

high density lipoprotein, which can remove cholesterol from cells and antheroma, offer protection and

are referred as good cholesterol.

The effect of ethanol leaf extract of Moringa oleifera on triacylglycerol concentration in mice showed a

non significant difference (p>0.05) in triacylglycerol in all the groups compared to the triacylglycerol

concentration of mice in group 6 (negative control). This showed that malaria had no effect on the

triacylglycerol concentration in the mice.But there was a non significant decrease (p>0.05) in

triacylglycerol concentration in group 2(45mg/kg body weight of the extract), group 3 (90mg/kg body

weight of the extract) and group 5 (5mg/kg body weight of the artesunate) when compared to group 1

(positive control) and these are consistent with the works of Ghasi et al. (2000) and Mehta et al.

(2003). Triacylglycerol (TAG) is a major component of very low density lipoprotein and chylomicrons

which play important role in metabolism as energy sources and transporters of dietary fats (Mehta et

al., 2003).

4.2 Conclusion

In conclusion, the results shown in this work indicate that ethanol leaf extract of Moringa oleifera

might have some antimalarial properties. The extract cleared parasitaemia in mice that were infected

with malaria. This research work also suggests that the ethanol leaf extract of Moringa oleifera

(Agbaji) has the potential or efficacy of boosting the haematological parameters. These help in the

protection of the liver, kidney and other vital organs from damages due to malaria parasitaemia. The

extract helped in ameliorating the adverse effect of malaria on the liver and kidney of the mice. The

203

extract was found to have non-significant effects on lipid metabolism. These findings support the

reports that Moringa oleifera commonly known as Agbaji is used for the local treatment of malaria

and is an important ingredient of polyherbal formulations marketed for the treatment of malaria and

other parasitic diseases. Therefore, leaf extract of Moringa oleifera should be encouraged as an

efficacious drug for malaria.

4.3 Suggestions for Further Research

1. Comparative analyses of antimalarial effect of ethanol leaf extract of Moringa oleifera singly

and in combination with another known antimalarial drugs should be carried out.

2. Comparative analyses of all the different parts of Moringa oleifera tree should be analysed in

order to determine the part with highest antimalaria potency.

3. Effect of the ethanol leaf extract of Moringa oleifera on protein and carbohydrate metabolism

should be carried out.

4. A thorough research on whether the ethanol leaf extract of Moringa oleifera is best for

chemotherapy and chemoprophylaxis of malaria should be assayed.

5. The plant extract should be used in a more purified form.

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APPENDICES

213

Appendix I: Calibrated Activity of AST in Serum

ABSORBANCE U/L

0.020 7

0.030 10

0.040 13

0.050 16

0.060 19

0.070 23

0.080 27

0.090 31

0.0100 36

Appendix II: Calibrated Activity of ALT in Serum

ABSORBANCE U/L

0.025 4

0.050 8

214

0.075 12

0.100 17

0.125 21

0.150 25

0.175 29

0.200 34

0.225 39

0.250 43

Appendix III: Descriptive and Multiple Comparison Table of Percentage Parasitaemia

215

Group 1 (Positive control)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

3 Days after innoculation 4 1.5500 .76811 .38406

Day 3 of Treatments 4 2.9750 .41932 .20966

Day 5 of Treatment 4 6.8500 1.70196 .85098

Day 28 Post-treatment 4 8.1750 .35000 .17500

Total 16 4.8875 2.93459 .73365

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Days (J) Days Mean Difference (I-J) Std. Error Sig.

LSD 3 Days after innoculation Day 3 of Treatments -1.42500 .68784 .061

Day 5 of Treatment -5.30000* .68784 .000

Day 28 Post-treatment -6.62500* .68784 .000

Day 3 of Treatments 3 Days after innoculation 1.42500 .68784 .061

Day 5 of Treatment -3.87500* .68784 .000

Day 28 Post-treatment -5.20000* .68784 .000

Day 5 of Treatment 3 Days after innoculation 5.30000* .68784 .000

Day 3 of Treatments 3.87500* .68784 .000

Day 28 Post-treatment -1.32500 .68784 .078

Day 28 Post-treatment 3 Days after innoculation 6.62500* .68784 .000

216

Day 3 of Treatments 5.20000* .68784 .000

Day 5 of Treatment 1.32500 .68784 .078

*. The mean difference is significant at the 0.05 level.

Oneway (Group 2: 45mg/kg b.w. of Extract)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

3 Days after innoculation 4 1.6750 1.22848 .61424

Day 3 of Treatments 4 3.3000 1.42829 .71414

Day 5 of Treatment 4 3.3750 1.14710 .57355

Day 28 Post-treatment 4 4.5500 .71414 .35707

Total 16 3.2250 1.48032 .37008

Post Hoc Tests

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Days (J) Days Mean

Difference (I-J) Std. Error Sig.

LSD 3 Days after innoculation Day 3 of Treatments -1.62500 .81968 .071

Day 5 of Treatment -1.70000 .81968 .060

Day 28 Post-treatment -2.87500* .81968 .004

Day 3 of Treatments 3 Days after innoculation 1.62500 .81968 .071

Day 5 of Treatment -.07500 .81968 .929

217

Day 28 Post-treatment -1.25000 .81968 .153

Day 5 of Treatment 3 Days after innoculation 1.70000 .81968 .060

Day 3 of Treatments .07500 .81968 .929

Day 28 Post-treatment -1.17500 .81968 .177

Day 28 Post-treatment 3 Days after innoculation 2.87500* .81968 .004

Day 3 of Treatments 1.25000 .81968 .153

Day 5 of Treatment 1.17500 .81968 .177

*. The mean difference is significant at the 0.05 level.

Oneway (Group 3: 90mg/kg b.w. Extract)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

3 Days after innoculation 4 1.5750 .72744 .36372

Day 3 of Treatments 4 2.2500 .64031 .32016

Day 5 of Treatment 4 1.5500 .77675 .38837

Day 28 Post-treatment 4 1.4500 1.05040 .52520

Total 16 1.7063 .79789 .19947

Post Hoc Tests

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Days (J) Days Mean Difference (I-J) Std. Error Sig.

218

LSD 3 Days after innoculation Day 3 of Treatments -.67500 .57509 .263

Day 5 of Treatment .02500 .57509 .966

Day 28 Post-treatment .12500 .57509 .832

Day 3 of Treatments 3 Days after innoculation .67500 .57509 .263

Day 5 of Treatment .70000 .57509 .247

Day 28 Post-treatment .80000 .57509 .189

Day 5 of Treatment 3 Days after innoculation -.02500 .57509 .966

Day 3 of Treatments -.70000 .57509 .247

Day 28 Post-treatment .10000 .57509 .865

Day 28 Post-treatment 3 Days after innoculation -.12500 .57509 .832

Day 3 of Treatments -.80000 .57509 .189

Day 5 of Treatment -.10000 .57509 .865

Oneway (Group 4: 180mg/kg b.w. of Extract)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

3 Days after innoculation 4 1.1250 .51881 .25941

Day 3 of Treatments 4 .7750 .51881 .25941

Day 5 of Treatment 4 .4750 .28723 .14361

Day 28 Post-treatment 4 .0025 .00500 .00250

Total 16 .5944 .55242 .13811

Post Hoc Tests

219

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Days (J) Days Mean

Difference (I-J) Std. Error Sig.

LSD 3 Days after innoculation Day 3 of Treatments .35000 .27858 .233

Day 5 of Treatment .65000* .27858 .038

Day 28 Post-treatment 1.12250* .27858 .002

Day 3 of Treatments 3 Days after innoculation -.35000 .27858 .233

Day 5 of Treatment .30000 .27858 .303

Day 28 Post-treatment .77250* .27858 .017

Day 5 of Treatment 3 Days after innoculation -.65000* .27858 .038

Day 3 of Treatments -.30000 .27858 .303

Day 28 Post-treatment .47250 .27858 .116

Day 28 Post-treatment 3 Days after innoculation -1.12250* .27858 .002

Day 3 of Treatments -.77250* .27858 .017

Day 5 of Treatment -.47250 .27858 .116

*. The mean difference is significant at the 0.05 level.

220

Oneway (Group 5: 5mg/kg b.w of Artesunate)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

3 Days after innoculation 4 .6750 .12583 .06292

Day 3 of Treatments 4 .6000 .24495 .12247

Day 5 of Treatment 4 .4000 .21602 .10801

Day 28 Post-treatment 4 .0150 .03000 .01500

Total 16 .4225 .30741 .07685

Post Hoc Tests

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Days (J) Days Mean Difference (I-J) Std. Error Sig.

LSD 3 Days after innoculation Day 3 of Treatments .07500 .12420 .557

Day 5 of Treatment .27500* .12420 .047

Day 28 Post-treatment .66000* .12420 .000

Day 3 of Treatments 3 Days after innoculation -.07500 .12420 .557

Day 5 of Treatment .20000 .12420 .133

Day 28 Post-treatment .58500* .12420 .001

Day 5 of Treatment 3 Days after innoculation -.27500* .12420 .047

Day 3 of Treatments -.20000 .12420 .133

Day 28 Post-treatment .38500* .12420 .009

221

Day 28 Post-treatment 3 Days after innoculation -.66000* .12420 .000

Day 3 of Treatments -.58500* .12420 .001

Day 5 of Treatment -.38500* .12420 .009

*. The mean difference is significant at the 0.05 level.

Oneway (Group 6: Negative Control=5mg/kg of Distilled Water)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

3 Days after innoculation 4 .0000 .00000 .00000

Day 3 of Treatments 4 .0000 .00000 .00000

Day 5 of Treatment 4 .0000 .00000 .00000

Day 28 Post-treatment 4 .0000 .00000 .00000

Total 16 .0000 .00000 .00000

Post Hoc Tests

Oneway (3 Days After Innoculation)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

Group 1 (Positive Control) 4 1.5500 .76811 .38406

222

Group 2 (45 mg/kg Extract) 4 1.6750 1.22848 .61424

Group 3 (90mg/kg of Extract) 4 1.5750 .72744 .36372

Group 4 (180mg/kg of Extract) 4 1.1250 .51881 .25941

Group 5 (5mg/kg of Artesunate) 4 .6750 .12583 .06292

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 .0000 .00000 .00000

Total 24 1.1000 .86828 .17724

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Group (J) Group

Mean Difference

(I-J)

Std.

Error Sig.

95% Confidence Interval

Lower

Bound

Upper

Bound

LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) -.12500 .49272 .803 -1.1602 .9102

Group 3 (90mg/kg of Extract) -.02500 .49272 .960 -1.0602 1.0102

Group 4 (180mg/kg of Extract) .42500 .49272 .400 -.6102 1.4602

Group 5 (5mg/kg of Artesunate) .87500 .49272 .093 -.1602 1.9102

Group 6 (Negative

Control=5mg/kg of Distilled Water)

1.55000* .49272 .006 .5148 2.5852

Group 2 (45

mg/kg Extract)

Group 1 (Positive Control) .12500 .49272 .803 -.9102 1.1602

Group 3 (90mg/kg of Extract) .10000 .49272 .841 -.9352 1.1352

Group 4 (180mg/kg of Extract) .55000 .49272 .279 -.4852 1.5852

Group 5 (5mg/kg of Artesunate) 1.00000 .49272 .057 -.0352 2.0352

Group 6 (Negative

Control=5mg/kg of Distilled Water)

1.67500* .49272 .003 .6398 2.7102

223

Group 3

(90mg/kg of

Extract)

Group 1 (Positive Control) .02500 .49272 .960 -1.0102 1.0602

Group 2 (45 mg/kg Extract) -.10000 .49272 .841 -1.1352 .9352

Group 4 (180mg/kg of Extract) .45000 .49272 .373 -.5852 1.4852

Group 5 (5mg/kg of Artesunate) .90000 .49272 .084 -.1352 1.9352

Group 6 (Negative

Control=5mg/kg of Distilled Water)

1.57500* .49272 .005 .5398 2.6102

Group 4

(180mg/kg of

Extract)

Group 1 (Positive Control) -.42500 .49272 .400 -1.4602 .6102

Group 2 (45 mg/kg Extract) -.55000 .49272 .279 -1.5852 .4852

Group 3 (90mg/kg of Extract) -.45000 .49272 .373 -1.4852 .5852

Group 5 (5mg/kg of Artesunate) .45000 .49272 .373 -.5852 1.4852

Group 6 (Negative

Control=5mg/kg of Distilled Water)

1.12500* .49272 .035 .0898 2.1602

Group 5 (5mg/kg

of Artesunate)

Group 1 (Positive Control) -.87500 .49272 .093 -1.9102 .1602

Group 2 (45 mg/kg Extract) -1.00000 .49272 .057 -2.0352 .0352

Group 3 (90mg/kg of Extract) -.90000 .49272 .084 -1.9352 .1352

Group 4 (180mg/kg of Extract) -.45000 .49272 .373 -1.4852 .5852

Group 6 (Negative

Control=5mg/kg of Distilled Water)

.67500 .49272 .188 -.3602 1.7102

Group 6

(Negative

Control=5mg/kg

of Distilled Water)

Group 1 (Positive Control) -1.55000* .49272 .006 -2.5852 -.5148

Group 2 (45 mg/kg Extract) -1.67500* .49272 .003 -2.7102 -.6398

Group 3 (90mg/kg of Extract) -1.57500* .49272 .005 -2.6102 -.5398

Group 4 (180mg/kg of Extract) -1.12500* .49272 .035 -2.1602 -.0898

Group 5 (5mg/kg of Artesunate) -.67500 .49272 .188 -1.7102 .3602

*. The mean difference is significant at the 0.05 level.

224

Oneway (Day 3 of Treatment)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

Group 1 (Positive Control) 4 2.9750 .41932 .20966

Group 2 (45 mg/kg Extract) 4 3.3000 1.42829 .71414

Group 3 (90mg/kg of Extract) 4 2.2500 .64031 .32016

Group 4 (180mg/kg of Extract) 4 .7750 .51881 .25941

Group 5 (5mg/kg of Artesunate) 4 .6000 .24495 .12247

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 .0000 .00000 .00000

Total 24 1.6500 1.42310 .29049

Descriptives

% Parasitaemia

95% Confidence Interval for Mean

Minimum Maximum Lower Bound Upper Bound

Group 1 (Positive Control) 2.3078 3.6422 2.70 3.60

225

Group 2 (45 mg/kg Extract) 1.0273 5.5727 2.20 5.20

Group 3 (90mg/kg of Extract) 1.2311 3.2689 1.80 3.20

Group 4 (180mg/kg of Extract) -.0505 1.6005 .40 1.50

Group 5 (5mg/kg of Artesunate) .2102 .9898 .40 .90

Group 6 (Negative Control=5mg/kg of Distilled Water) .0000 .0000 .00 .00

Total 1.0491 2.2509 .00 5.20

226

227

Post Hoc Tests

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Group (J) Group

Mean Difference (I-

J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) -.32500 .49624 .521 -1.3676 .7176

Group 3 (90mg/kg of Extract) .72500 .49624 .161 -.3176 1.7676

Group 4 (180mg/kg of Extract) 2.20000* .49624 .000 1.1574 3.2426

Group 5 (5mg/kg of Artesunate) 2.37500* .49624 .000 1.3324 3.4176

Group 6 (Negative Control=5mg/kg

of Distilled Water)

2.97500* .49624 .000 1.9324 4.0176

Group 2 (45 mg/kg Extract) Group 1 (Positive Control) .32500 .49624 .521 -.7176 1.3676

Group 3 (90mg/kg of Extract) 1.05000* .49624 .049 .0074 2.0926

Group 4 (180mg/kg of Extract) 2.52500* .49624 .000 1.4824 3.5676

Group 5 (5mg/kg of Artesunate) 2.70000* .49624 .000 1.6574 3.7426

228

Group 6 (Negative Control=5mg/kg

of Distilled Water)

3.30000* .49624 .000 2.2574 4.3426

Group 3 (90mg/kg of Extract) Group 1 (Positive Control) -.72500 .49624 .161 -1.7676 .3176

Group 2 (45 mg/kg Extract) -1.05000* .49624 .049 -2.0926 -.0074

Group 4 (180mg/kg of Extract) 1.47500* .49624 .008 .4324 2.5176

Group 5 (5mg/kg of Artesunate) 1.65000* .49624 .004 .6074 2.6926

Group 6 (Negative Control=5mg/kg

of Distilled Water)

2.25000* .49624 .000 1.2074 3.2926

Group 4 (180mg/kg of Extract) Group 1 (Positive Control) -2.20000* .49624 .000 -3.2426 -1.1574

Group 2 (45 mg/kg Extract) -2.52500* .49624 .000 -3.5676 -1.4824

Group 3 (90mg/kg of Extract) -1.47500* .49624 .008 -2.5176 -.4324

Group 5 (5mg/kg of Artesunate) .17500 .49624 .728 -.8676 1.2176

Group 6 (Negative Control=5mg/kg

of Distilled Water)

.77500 .49624 .136 -.2676 1.8176

Group 5 (5mg/kg of Artesunate) Group 1 (Positive Control) -2.37500* .49624 .000 -3.4176 -1.3324

Group 2 (45 mg/kg Extract) -2.70000* .49624 .000 -3.7426 -1.6574

Group 3 (90mg/kg of Extract) -1.65000* .49624 .004 -2.6926 -.6074

Group 4 (180mg/kg of Extract) -.17500 .49624 .728 -1.2176 .8676

229

Group 6 (Negative Control=5mg/kg

of Distilled Water)

.60000 .49624 .242 -.4426 1.6426

Group 6 (Negative

Control=5mg/kg of Distilled

Water)

Group 1 (Positive Control) -2.97500* .49624 .000 -4.0176 -1.9324

Group 2 (45 mg/kg Extract) -3.30000* .49624 .000 -4.3426 -2.2574

Group 3 (90mg/kg of Extract) -2.25000* .49624 .000 -3.2926 -1.2074

Group 4 (180mg/kg of Extract) -.77500 .49624 .136 -1.8176 .2676

Group 5 (5mg/kg of Artesunate) -.60000 .49624 .242 -1.6426 .4426

*. The mean difference is significant at the 0.05 level.

230

Oneway (Day 3 of Treatment)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

Group 1 (Positive Control) 4 6.8500 1.70196 .85098

Group 2 (45 mg/kg Extract) 4 3.3750 1.14710 .57355

Group 3 (90mg/kg of Extract) 4 1.5500 .77675 .38837

Group 4 (180mg/kg of Extract) 4 .4750 .28723 .14361

Group 5 (5mg/kg of Artesunate) 4 .4000 .21602 .10801

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 .0000 .00000 .00000

Total 24 2.1083 2.57546 .52571

Oneway (Day 28 of Post-Treatment)

Descriptives

% Parasitaemia

N Mean Std. Deviation Std. Error

Group 1 (Positive Control) 4 8.1750 .35000 .17500

Group 2 (45 mg/kg Extract) 4 4.5500 .71414 .35707

Group 3 (90mg/kg of Extract) 4 1.4500 1.05040 .52520

Group 4 (180mg/kg of Extract) 4 .0025 .00500 .00250

Group 5 (5mg/kg of Artesunate) 4 .0150 .03000 .01500

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 .0000 .00000 .00000

Total 24 2.3654 3.15863 .64475

231

Post Hoc Tests

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Group (J) Group

Mean Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) 3.47500* .64194 .000 2.1263 4.8237

Group 3 (90mg/kg of Extract) 5.30000* .64194 .000 3.9513 6.6487

Group 4 (180mg/kg of Extract) 6.37500* .64194 .000 5.0263 7.7237

Group 5 (5mg/kg of Artesunate) 6.45000* .64194 .000 5.1013 7.7987

Group 6 (Negative Control=5mg/kg of Distilled Water) 6.85000* .64194 .000 5.5013 8.1987

Group 2 (45 mg/kg Extract) Group 1 (Positive Control) -3.47500* .64194 .000 -4.8237 -2.1263

Group 3 (90mg/kg of Extract) 1.82500* .64194 .011 .4763 3.1737

Group 4 (180mg/kg of Extract) 2.90000* .64194 .000 1.5513 4.2487

Group 5 (5mg/kg of Artesunate) 2.97500* .64194 .000 1.6263 4.3237

Group 6 (Negative Control=5mg/kg of Distilled Water) 3.37500* .64194 .000 2.0263 4.7237

232

Group 3 (90mg/kg of Extract) Group 1 (Positive Control) -5.30000* .64194 .000 -6.6487 -3.9513

Group 2 (45 mg/kg Extract) -1.82500* .64194 .011 -3.1737 -.4763

Group 4 (180mg/kg of Extract) 1.07500 .64194 .111 -.2737 2.4237

Group 5 (5mg/kg of Artesunate) 1.15000 .64194 .090 -.1987 2.4987

Group 6 (Negative Control=5mg/kg of Distilled Water) 1.55000* .64194 .027 .2013 2.8987

Group 4 (180mg/kg of Extract) Group 1 (Positive Control) -6.37500* .64194 .000 -7.7237 -5.0263

Group 2 (45 mg/kg Extract) -2.90000* .64194 .000 -4.2487 -1.5513

Group 3 (90mg/kg of Extract) -1.07500 .64194 .111 -2.4237 .2737

Group 5 (5mg/kg of Artesunate) .07500 .64194 .908 -1.2737 1.4237

Group 6 (Negative Control=5mg/kg of Distilled Water) .47500 .64194 .469 -.8737 1.8237

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -6.45000* .64194 .000 -7.7987 -5.1013

Group 2 (45 mg/kg Extract) -2.97500* .64194 .000 -4.3237 -1.6263

Group 3 (90mg/kg of Extract) -1.15000 .64194 .090 -2.4987 .1987

Group 4 (180mg/kg of Extract) -.07500 .64194 .908 -1.4237 1.2737

Group 6 (Negative Control=5mg/kg of Distilled Water) .40000 .64194 .541 -.9487 1.7487

Group 6 (Negative

Control=5mg/kg of Distilled

Group 1 (Positive Control) -6.85000* .64194 .000 -8.1987 -5.5013

Group 2 (45 mg/kg Extract) -3.37500* .64194 .000 -4.7237 -2.0263

233

Water) Group 3 (90mg/kg of Extract) -1.55000* .64194 .027 -2.8987 -.2013

Group 4 (180mg/kg of Extract) -.47500 .64194 .469 -1.8237 .8737

Group 5 (5mg/kg of Artesunate) -.40000 .64194 .541 -1.7487 .9487

*. The mean difference is significant at the 0.05 level.

Post Hoc Tests

Multiple Comparisons

Dependent Variable:% Parasitaemia

(I) Group (J) Group

Mean Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) 3.62500* .38043 .000 2.8257 4.4243

Group 3 (90mg/kg of Extract) 6.72500* .38043 .000 5.9257 7.5243

Group 4 (180mg/kg of Extract) 8.17250* .38043 .000 7.3732 8.9718

Group 5 (5mg/kg of Artesunate) 8.16000* .38043 .000 7.3607 8.9593

Group 6 (Negative Control=5mg/kg of Distilled Water) 8.17500* .38043 .000 7.3757 8.9743

Group 2 (45 mg/kg Extract) Group 1 (Positive Control) -3.62500* .38043 .000 -4.4243 -2.8257

Group 3 (90mg/kg of Extract) 3.10000* .38043 .000 2.3007 3.8993

234

Group 4 (180mg/kg of Extract) 4.54750* .38043 .000 3.7482 5.3468

Group 5 (5mg/kg of Artesunate) 4.53500* .38043 .000 3.7357 5.3343

Group 6 (Negative Control=5mg/kg of Distilled Water) 4.55000* .38043 .000 3.7507 5.3493

Group 3 (90mg/kg of Extract) Group 1 (Positive Control) -6.72500* .38043 .000 -7.5243 -5.9257

Group 2 (45 mg/kg Extract) -3.10000* .38043 .000 -3.8993 -2.3007

Group 4 (180mg/kg of Extract) 1.44750* .38043 .001 .6482 2.2468

Group 5 (5mg/kg of Artesunate) 1.43500* .38043 .001 .6357 2.2343

Group 6 (Negative Control=5mg/kg of Distilled Water) 1.45000* .38043 .001 .6507 2.2493

Group 4 (180mg/kg of Extract) Group 1 (Positive Control) -8.17250* .38043 .000 -8.9718 -7.3732

Group 2 (45 mg/kg Extract) -4.54750* .38043 .000 -5.3468 -3.7482

Group 3 (90mg/kg of Extract) -1.44750* .38043 .001 -2.2468 -.6482

Group 5 (5mg/kg of Artesunate) -.01250 .38043 .974 -.8118 .7868

Group 6 (Negative Control=5mg/kg of Distilled Water) .00250 .38043 .995 -.7968 .8018

Group 5 (5mg/kg of Artesunate) Group 1 (Positive Control) -8.16000* .38043 .000 -8.9593 -7.3607

Group 2 (45 mg/kg Extract) -4.53500* .38043 .000 -5.3343 -3.7357

Group 3 (90mg/kg of Extract) -1.43500* .38043 .001 -2.2343 -.6357

Group 4 (180mg/kg of Extract) .01250 .38043 .974 -.7868 .8118

235

Group 6 (Negative Control=5mg/kg of Distilled Water) .01500 .38043 .969 -.7843 .8143

Group 6 (Negative

Control=5mg/kg of Distilled

Water)

Group 1 (Positive Control) -8.17500* .38043 .000 -8.9743 -7.3757

Group 2 (45 mg/kg Extract) -4.55000* .38043 .000 -5.3493 -3.7507

Group 3 (90mg/kg of Extract) -1.45000* .38043 .001 -2.2493 -.6507

Group 4 (180mg/kg of Extract) -.00250 .38043 .995 -.8018 .7968

Group 5 (5mg/kg of Artesunate) -.01500 .38043 .969 -.8143 .7843

*. The mean difference is significant at the 0.05 level.

236

APPENDIX IV: Multiple Comparison of Biochemical Parameters

Descriptives

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum Maximum Lower Bound Upper Bound

ALT Group 1 (Positive Control) 4 42.5000 7.32575 3.66288 30.8431 54.1569 34.00 50.00

Group 2 (45 mg/kg Extract) 4 34.2500 2.50000 1.25000 30.2719 38.2281 31.00 37.00

Group 3 (90mg/kg of Extract) 4 27.0000 4.69042 2.34521 19.5365 34.4635 22.00 33.00

Group 4 (180mg/kg of Extract) 4 27.2500 3.20156 1.60078 22.1556 32.3444 24.00 30.00

Group 5 (5mg/kg of Artesunate) 4 26.2500 2.98608 1.49304 21.4985 31.0015 23.00 30.00

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 24.7500 2.87228 1.43614 20.1796 29.3204 23.00 29.00

Total 24 30.3333 7.38781 1.50803 27.2137 33.4529 22.00 50.00

AST Group 1 (Positive Control) 4 209.5000 8.58293 4.29146 195.8426 223.1574 199.00 220.00

Group 2 (45 mg/kg Extract) 4 207.0000 7.39369 3.69685 195.2350 218.7650 200.00 216.00

Group 3 (90mg/kg of Extract) 4 212.0000 8.32666 4.16333 198.7504 225.2496 202.00 222.00

Group 4 (180mg/kg of Extract) 4 205.0000 15.29706 7.64853 180.6590 229.3410 189.00 220.00

237

Group 5 (5mg/kg of Artesunate) 4 210.0000 4.08248 2.04124 203.5039 216.4961 205.00 215.00

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 207.0000 7.07107 3.53553 195.7484 218.2516 197.00 212.00

Total 24 208.4167 8.40247 1.71515 204.8686 211.9647 189.00 222.00

ALP Group 1 (Positive Control) 4 179.5000 12.55654 6.27827 159.5197 199.4803 169.00 197.00

Group 2 (45 mg/kg Extract) 4 147.2500 10.24288 5.12144 130.9513 163.5487 138.00 160.00

Group 3 (90mg/kg of Extract) 4 150.2500 1.70783 .85391 147.5325 152.9675 148.00 152.00

Group 4 (180mg/kg of Extract) 4 173.0000 19.40790 9.70395 142.1177 203.8823 149.00 192.00

Group 5 (5mg/kg of Artesunate) 4 154.5000 10.66146 5.33073 137.5352 171.4648 141.00 167.00

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 153.5000 12.66228 6.33114 133.3515 173.6485 139.00 168.00

Total 24 159.6667 16.50209 3.36847 152.6984 166.6349 138.00 197.00

TB Group 1 (Positive Control) 4 23.3000 .91287 .45644 21.8474 24.7526 22.20 24.10

Group 2 (45 mg/kg Extract) 4 22.2500 1.70783 .85391 19.5325 24.9675 20.00 24.00

Group 3 (90mg/kg of Extract) 4 19.3500 .99833 .49917 17.7614 20.9386 18.00 20.20

Group 4 (180mg/kg of Extract) 4 18.3000 .42426 .21213 17.6249 18.9751 17.90 18.80

Group 5 (5mg/kg of Artesunate) 4 16.2250 .80156 .40078 14.9495 17.5005 15.20 17.00

238

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 16.1500 .86987 .43493 14.7658 17.5342 15.00 17.00

Total 24 19.2625 2.94350 .60084 18.0196 20.5054 15.00 24.10

Creati

nine

Group 1 (Positive Control) 4 81.2500 2.98608 1.49304 76.4985 86.0015 78.00 85.00

Group 2 (45 mg/kg Extract) 4 77.7500 1.70783 .85391 75.0325 80.4675 76.00 80.00

Group 3 (90mg/kg of Extract) 4 75.2500 2.50000 1.25000 71.2719 79.2281 72.00 78.00

Group 4 (180mg/kg of Extract) 4 77.7500 1.25831 .62915 75.7478 79.7522 76.00 79.00

Group 5 (5mg/kg of Artesunate) 4 72.7500 .95743 .47871 71.2265 74.2735 72.00 74.00

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 72.5000 2.08167 1.04083 69.1876 75.8124 70.00 75.00

Total 24 76.2083 3.62334 .73961 74.6783 77.7383 70.00 85.00

Urea Group 1 (Positive Control) 4 5.9250 .17078 .08539 5.6532 6.1968 5.70 6.10

Group 2 (45 mg/kg Extract) 4 5.6750 .20616 .10308 5.3470 6.0030 5.40 5.90

Group 3 (90mg/kg of Extract) 4 5.8500 .05774 .02887 5.7581 5.9419 5.80 5.90

Group 4 (180mg/kg of Extract) 4 5.7250 .29861 .14930 5.2498 6.2002 5.40 6.10

Group 5 (5mg/kg of Artesunate) 4 6.0750 .09574 .04787 5.9227 6.2273 6.00 6.20

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 6.2000 .49666 .24833 5.4097 6.9903 5.80 6.90

239

Total 24 5.9083 .30060 .06136 5.7814 6.0353 5.40 6.90

Choles

terol

Group 1 (Positive Control) 4 3.1900 .29178 .14589 2.7257 3.6543 2.76 3.41

Group 2 (45 mg/kg Extract) 4 2.9950 .42194 .21097 2.3236 3.6664 2.40 3.30

Group 3 (90mg/kg of Extract) 4 3.1125 .34846 .17423 2.5580 3.6670 2.59 3.30

Group 4 (180mg/kg of Extract) 4 2.9050 .36014 .18007 2.3319 3.4781 2.42 3.28

Group 5 (5mg/kg of Artesunate) 4 2.9975 .53369 .26684 2.1483 3.8467 2.40 3.63

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 3.1775 .23186 .11593 2.8086 3.5464 2.83 3.30

Total 24 3.0629 .35021 .07149 2.9150 3.2108 2.40 3.63

HDL Group 1 (Positive Control) 4 .8300 .15033 .07517 .5908 1.0692 .68 1.00

Group 2 (45 mg/kg Extract) 4 .8975 .27220 .13610 .4644 1.3306 .65 1.21

Group 3 (90mg/kg of Extract) 4 .8775 .22292 .11146 .5228 1.2322 .55 1.04

Group 4 (180mg/kg of Extract) 4 .9275 .18857 .09428 .6274 1.2276 .66 1.09

Group 5 (5mg/kg of Artesunate) 4 .7450 .11269 .05635 .5657 .9243 .66 .91

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 .7375 .10046 .05023 .5777 .8973 .65 .88

Total 24 .8358 .17959 .03666 .7600 .9117 .55 1.21

LDL Group 1 (Positive Control) 4 1.6900 .40702 .20351 1.0423 2.3377 1.13 2.05

240

Group 2 (45 mg/kg Extract) 4 1.4800 .60083 .30042 .5239 2.4361 .59 1.91

Group 3 (90mg/kg of Extract) 4 1.6000 .46000 .23000 .8680 2.3320 .95 2.03

Group 4 (180mg/kg of Extract) 4 1.2025 .63892 .31946 .1858 2.2192 .47 2.02

Group 5 (5mg/kg of Artesunate) 4 1.6100 .47672 .23836 .8514 2.3686 1.11 2.24

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 1.7650 .30183 .15091 1.2847 2.2453 1.32 1.99

Total 24 1.5579 .47473 .09690 1.3575 1.7584 .47 2.24

TAG Group 1 (Positive Control) 4 1.4775 .10751 .05375 1.3064 1.6486 1.39 1.61

Group 2 (45 mg/kg Extract) 4 1.3600 .16912 .08456 1.0909 1.6291 1.24 1.61

Group 3 (90mg/kg of Extract) 4 1.3975 .14009 .07004 1.1746 1.6204 1.24 1.53

Group 4 (180mg/kg of Extract) 4 1.5300 .25652 .12826 1.1218 1.9382 1.33 1.90

Group 5 (5mg/kg of Artesunate) 4 1.4100 .03367 .01683 1.3564 1.4636 1.39 1.46

Group 6 (Negative Control=5mg/kg of

Distilled Water)

4 1.4900 .09201 .04601 1.3436 1.6364 1.39 1.61

Total 24 1.4442 .14590 .02978 1.3826 1.5058 1.24 1.90

Post Hoc Tests

241

Multiple Comparisons

Dependent

Variable

(I) Group (J) Group

Mean Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

ALT LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) 8.25000* 3.01846 .014 1.9084 14.5916

Group 3 (90mg/kg of Extract) 15.50000* 3.01846 .000 9.1584 21.8416

Group 4 (180mg/kg of Extract) 15.25000* 3.01846 .000 8.9084 21.5916

Group 5 (5mg/kg of Artesunate) 16.25000* 3.01846 .000 9.9084 22.5916

Group 6 (Negative Control=5mg/kg of

Distilled Water)

17.75000* 3.01846 .000 11.4084 24.0916

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -8.25000* 3.01846 .014 -14.5916 -1.9084

Group 3 (90mg/kg of Extract) 7.25000* 3.01846 .027 .9084 13.5916

Group 4 (180mg/kg of Extract) 7.00000* 3.01846 .032 .6584 13.3416

Group 5 (5mg/kg of Artesunate) 8.00000* 3.01846 .016 1.6584 14.3416

Group 6 (Negative Control=5mg/kg of

Distilled Water)

9.50000* 3.01846 .006 3.1584 15.8416

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -15.50000* 3.01846 .000 -21.8416 -9.1584

Group 2 (45 mg/kg Extract) -7.25000* 3.01846 .027 -13.5916 -.9084

Group 4 (180mg/kg of Extract) -.25000 3.01846 .935 -6.5916 6.0916

242

Group 5 (5mg/kg of Artesunate) .75000 3.01846 .807 -5.5916 7.0916

Group 6 (Negative Control=5mg/kg of

Distilled Water)

2.25000 3.01846 .466 -4.0916 8.5916

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -15.25000* 3.01846 .000 -21.5916 -8.9084

Group 2 (45 mg/kg Extract) -7.00000* 3.01846 .032 -13.3416 -.6584

Group 3 (90mg/kg of Extract) .25000 3.01846 .935 -6.0916 6.5916

Group 5 (5mg/kg of Artesunate) 1.00000 3.01846 .744 -5.3416 7.3416

Group 6 (Negative Control=5mg/kg of

Distilled Water)

2.50000 3.01846 .418 -3.8416 8.8416

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -16.25000* 3.01846 .000 -22.5916 -9.9084

Group 2 (45 mg/kg Extract) -8.00000* 3.01846 .016 -14.3416 -1.6584

Group 3 (90mg/kg of Extract) -.75000 3.01846 .807 -7.0916 5.5916

Group 4 (180mg/kg of Extract) -1.00000 3.01846 .744 -7.3416 5.3416

Group 6 (Negative Control=5mg/kg of

Distilled Water)

1.50000 3.01846 .625 -4.8416 7.8416

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -17.75000* 3.01846 .000 -24.0916 -11.4084

Group 2 (45 mg/kg Extract) -9.50000* 3.01846 .006 -15.8416 -3.1584

Group 3 (90mg/kg of Extract) -2.25000 3.01846 .466 -8.5916 4.0916

243

Group 4 (180mg/kg of Extract) -2.50000 3.01846 .418 -8.8416 3.8416

Group 5 (5mg/kg of Artesunate) -1.50000 3.01846 .625 -7.8416 4.8416

AST LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) 2.50000 6.44420 .703 -11.0388 16.0388

Group 3 (90mg/kg of Extract) -2.50000 6.44420 .703 -16.0388 11.0388

Group 4 (180mg/kg of Extract) 4.50000 6.44420 .494 -9.0388 18.0388

Group 5 (5mg/kg of Artesunate) -.50000 6.44420 .939 -14.0388 13.0388

Group 6 (Negative Control=5mg/kg of

Distilled Water)

2.50000 6.44420 .703 -11.0388 16.0388

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -2.50000 6.44420 .703 -16.0388 11.0388

Group 3 (90mg/kg of Extract) -5.00000 6.44420 .448 -18.5388 8.5388

Group 4 (180mg/kg of Extract) 2.00000 6.44420 .760 -11.5388 15.5388

Group 5 (5mg/kg of Artesunate) -3.00000 6.44420 .647 -16.5388 10.5388

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.00000 6.44420 1.000 -13.5388 13.5388

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) 2.50000 6.44420 .703 -11.0388 16.0388

Group 2 (45 mg/kg Extract) 5.00000 6.44420 .448 -8.5388 18.5388

Group 4 (180mg/kg of Extract) 7.00000 6.44420 .292 -6.5388 20.5388

Group 5 (5mg/kg of Artesunate) 2.00000 6.44420 .760 -11.5388 15.5388

244

Group 6 (Negative Control=5mg/kg of

Distilled Water)

5.00000 6.44420 .448 -8.5388 18.5388

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -4.50000 6.44420 .494 -18.0388 9.0388

Group 2 (45 mg/kg Extract) -2.00000 6.44420 .760 -15.5388 11.5388

Group 3 (90mg/kg of Extract) -7.00000 6.44420 .292 -20.5388 6.5388

Group 5 (5mg/kg of Artesunate) -5.00000 6.44420 .448 -18.5388 8.5388

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-2.00000 6.44420 .760 -15.5388 11.5388

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) .50000 6.44420 .939 -13.0388 14.0388

Group 2 (45 mg/kg Extract) 3.00000 6.44420 .647 -10.5388 16.5388

Group 3 (90mg/kg of Extract) -2.00000 6.44420 .760 -15.5388 11.5388

Group 4 (180mg/kg of Extract) 5.00000 6.44420 .448 -8.5388 18.5388

Group 6 (Negative Control=5mg/kg of

Distilled Water)

3.00000 6.44420 .647 -10.5388 16.5388

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -2.50000 6.44420 .703 -16.0388 11.0388

Group 2 (45 mg/kg Extract) .00000 6.44420 1.000 -13.5388 13.5388

Group 3 (90mg/kg of Extract) -5.00000 6.44420 .448 -18.5388 8.5388

Group 4 (180mg/kg of Extract) 2.00000 6.44420 .760 -11.5388 15.5388

245

Group 5 (5mg/kg of Artesunate) -3.00000 6.44420 .647 -16.5388 10.5388

ALP LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) 32.25000* 8.73769 .002 13.8928 50.6072

Group 3 (90mg/kg of Extract) 29.25000* 8.73769 .004 10.8928 47.6072

Group 4 (180mg/kg of Extract) 6.50000 8.73769 .467 -11.8572 24.8572

Group 5 (5mg/kg of Artesunate) 25.00000* 8.73769 .010 6.6428 43.3572

Group 6 (Negative Control=5mg/kg of

Distilled Water)

26.00000* 8.73769 .008 7.6428 44.3572

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -32.25000* 8.73769 .002 -50.6072 -13.8928

Group 3 (90mg/kg of Extract) -3.00000 8.73769 .735 -21.3572 15.3572

Group 4 (180mg/kg of Extract) -25.75000* 8.73769 .009 -44.1072 -7.3928

Group 5 (5mg/kg of Artesunate) -7.25000 8.73769 .418 -25.6072 11.1072

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-6.25000 8.73769 .484 -24.6072 12.1072

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -29.25000* 8.73769 .004 -47.6072 -10.8928

Group 2 (45 mg/kg Extract) 3.00000 8.73769 .735 -15.3572 21.3572

Group 4 (180mg/kg of Extract) -22.75000* 8.73769 .018 -41.1072 -4.3928

Group 5 (5mg/kg of Artesunate) -4.25000 8.73769 .633 -22.6072 14.1072

246

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-3.25000 8.73769 .714 -21.6072 15.1072

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -6.50000 8.73769 .467 -24.8572 11.8572

Group 2 (45 mg/kg Extract) 25.75000* 8.73769 .009 7.3928 44.1072

Group 3 (90mg/kg of Extract) 22.75000* 8.73769 .018 4.3928 41.1072

Group 5 (5mg/kg of Artesunate) 18.50000* 8.73769 .048 .1428 36.8572

Group 6 (Negative Control=5mg/kg of

Distilled Water)

19.50000* 8.73769 .039 1.1428 37.8572

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -25.00000* 8.73769 .010 -43.3572 -6.6428

Group 2 (45 mg/kg Extract) 7.25000 8.73769 .418 -11.1072 25.6072

Group 3 (90mg/kg of Extract) 4.25000 8.73769 .633 -14.1072 22.6072

Group 4 (180mg/kg of Extract) -18.50000* 8.73769 .048 -36.8572 -.1428

Group 6 (Negative Control=5mg/kg of

Distilled Water)

1.00000 8.73769 .910 -17.3572 19.3572

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -26.00000* 8.73769 .008 -44.3572 -7.6428

Group 2 (45 mg/kg Extract) 6.25000 8.73769 .484 -12.1072 24.6072

Group 3 (90mg/kg of Extract) 3.25000 8.73769 .714 -15.1072 21.6072

Group 4 (180mg/kg of Extract) -19.50000* 8.73769 .039 -37.8572 -1.1428

247

Group 5 (5mg/kg of Artesunate) -1.00000 8.73769 .910 -19.3572 17.3572

TB LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) 1.05000 .72605 .165 -.4754 2.5754

Group 3 (90mg/kg of Extract) 3.95000* .72605 .000 2.4246 5.4754

Group 4 (180mg/kg of Extract) 5.00000* .72605 .000 3.4746 6.5254

Group 5 (5mg/kg of Artesunate) 7.07500* .72605 .000 5.5496 8.6004

Group 6 (Negative Control=5mg/kg of

Distilled Water)

7.15000* .72605 .000 5.6246 8.6754

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -1.05000 .72605 .165 -2.5754 .4754

Group 3 (90mg/kg of Extract) 2.90000* .72605 .001 1.3746 4.4254

Group 4 (180mg/kg of Extract) 3.95000* .72605 .000 2.4246 5.4754

Group 5 (5mg/kg of Artesunate) 6.02500* .72605 .000 4.4996 7.5504

Group 6 (Negative Control=5mg/kg of

Distilled Water)

6.10000* .72605 .000 4.5746 7.6254

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -3.95000* .72605 .000 -5.4754 -2.4246

Group 2 (45 mg/kg Extract) -2.90000* .72605 .001 -4.4254 -1.3746

Group 4 (180mg/kg of Extract) 1.05000 .72605 .165 -.4754 2.5754

Group 5 (5mg/kg of Artesunate) 3.12500* .72605 .000 1.5996 4.6504

248

Group 6 (Negative Control=5mg/kg of

Distilled Water)

3.20000* .72605 .000 1.6746 4.7254

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -5.00000* .72605 .000 -6.5254 -3.4746

Group 2 (45 mg/kg Extract) -3.95000* .72605 .000 -5.4754 -2.4246

Group 3 (90mg/kg of Extract) -1.05000 .72605 .165 -2.5754 .4754

Group 5 (5mg/kg of Artesunate) 2.07500* .72605 .010 .5496 3.6004

Group 6 (Negative Control=5mg/kg of

Distilled Water)

2.15000* .72605 .008 .6246 3.6754

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -7.07500* .72605 .000 -8.6004 -5.5496

Group 2 (45 mg/kg Extract) -6.02500* .72605 .000 -7.5504 -4.4996

Group 3 (90mg/kg of Extract) -3.12500* .72605 .000 -4.6504 -1.5996

Group 4 (180mg/kg of Extract) -2.07500* .72605 .010 -3.6004 -.5496

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.07500 .72605 .919 -1.4504 1.6004

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -7.15000* .72605 .000 -8.6754 -5.6246

Group 2 (45 mg/kg Extract) -6.10000* .72605 .000 -7.6254 -4.5746

Group 3 (90mg/kg of Extract) -3.20000* .72605 .000 -4.7254 -1.6746

Group 4 (180mg/kg of Extract) -2.15000* .72605 .008 -3.6754 -.6246

249

Group 5 (5mg/kg of Artesunate) -.07500 .72605 .919 -1.6004 1.4504

Creati-

nine

LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) 3.50000* 1.44097 .026 .4726 6.5274

Group 3 (90mg/kg of Extract) 6.00000* 1.44097 .001 2.9726 9.0274

Group 4 (180mg/kg of Extract) 3.50000* 1.44097 .026 .4726 6.5274

Group 5 (5mg/kg of Artesunate) 8.50000* 1.44097 .000 5.4726 11.5274

Group 6 (Negative Control=5mg/kg of

Distilled Water)

8.75000* 1.44097 .000 5.7226 11.7774

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -3.50000* 1.44097 .026 -6.5274 -.4726

Group 3 (90mg/kg of Extract) 2.50000 1.44097 .100 -.5274 5.5274

Group 4 (180mg/kg of Extract) .00000 1.44097 1.000 -3.0274 3.0274

Group 5 (5mg/kg of Artesunate) 5.00000* 1.44097 .003 1.9726 8.0274

Group 6 (Negative Control=5mg/kg of

Distilled Water)

5.25000* 1.44097 .002 2.2226 8.2774

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -6.00000* 1.44097 .001 -9.0274 -2.9726

Group 2 (45 mg/kg Extract) -2.50000 1.44097 .100 -5.5274 .5274

Group 4 (180mg/kg of Extract) -2.50000 1.44097 .100 -5.5274 .5274

Group 5 (5mg/kg of Artesunate) 2.50000 1.44097 .100 -.5274 5.5274

250

Group 6 (Negative Control=5mg/kg of

Distilled Water)

2.75000 1.44097 .072 -.2774 5.7774

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -3.50000* 1.44097 .026 -6.5274 -.4726

Group 2 (45 mg/kg Extract) .00000 1.44097 1.000 -3.0274 3.0274

Group 3 (90mg/kg of Extract) 2.50000 1.44097 .100 -.5274 5.5274

Group 5 (5mg/kg of Artesunate) 5.00000* 1.44097 .003 1.9726 8.0274

Group 6 (Negative Control=5mg/kg of

Distilled Water)

5.25000* 1.44097 .002 2.2226 8.2774

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -8.50000* 1.44097 .000 -11.5274 -5.4726

Group 2 (45 mg/kg Extract) -5.00000* 1.44097 .003 -8.0274 -1.9726

Group 3 (90mg/kg of Extract) -2.50000 1.44097 .100 -5.5274 .5274

Group 4 (180mg/kg of Extract) -5.00000* 1.44097 .003 -8.0274 -1.9726

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.25000 1.44097 .864 -2.7774 3.2774

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -8.75000* 1.44097 .000 -11.7774 -5.7226

Group 2 (45 mg/kg Extract) -5.25000* 1.44097 .002 -8.2774 -2.2226

Group 3 (90mg/kg of Extract) -2.75000 1.44097 .072 -5.7774 .2774

Group 4 (180mg/kg of Extract) -5.25000* 1.44097 .002 -8.2774 -2.2226

251

Group 5 (5mg/kg of Artesunate) -.25000 1.44097 .864 -3.2774 2.7774

Urea LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) .25000 .18708 .198 -.1430 .6430

Group 3 (90mg/kg of Extract) .07500 .18708 .693 -.3180 .4680

Group 4 (180mg/kg of Extract) .20000 .18708 .299 -.1930 .5930

Group 5 (5mg/kg of Artesunate) -.15000 .18708 .433 -.5430 .2430

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.27500 .18708 .159 -.6680 .1180

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.25000 .18708 .198 -.6430 .1430

Group 3 (90mg/kg of Extract) -.17500 .18708 .362 -.5680 .2180

Group 4 (180mg/kg of Extract) -.05000 .18708 .792 -.4430 .3430

Group 5 (5mg/kg of Artesunate) -.40000* .18708 .046 -.7930 -.0070

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.52500* .18708 .012 -.9180 -.1320

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -.07500 .18708 .693 -.4680 .3180

Group 2 (45 mg/kg Extract) .17500 .18708 .362 -.2180 .5680

Group 4 (180mg/kg of Extract) .12500 .18708 .513 -.2680 .5180

Group 5 (5mg/kg of Artesunate) -.22500 .18708 .245 -.6180 .1680

252

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.35000 .18708 .078 -.7430 .0430

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -.20000 .18708 .299 -.5930 .1930

Group 2 (45 mg/kg Extract) .05000 .18708 .792 -.3430 .4430

Group 3 (90mg/kg of Extract) -.12500 .18708 .513 -.5180 .2680

Group 5 (5mg/kg of Artesunate) -.35000 .18708 .078 -.7430 .0430

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.47500* .18708 .021 -.8680 -.0820

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) .15000 .18708 .433 -.2430 .5430

Group 2 (45 mg/kg Extract) .40000* .18708 .046 .0070 .7930

Group 3 (90mg/kg of Extract) .22500 .18708 .245 -.1680 .6180

Group 4 (180mg/kg of Extract) .35000 .18708 .078 -.0430 .7430

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.12500 .18708 .513 -.5180 .2680

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) .27500 .18708 .159 -.1180 .6680

Group 2 (45 mg/kg Extract) .52500* .18708 .012 .1320 .9180

Group 3 (90mg/kg of Extract) .35000 .18708 .078 -.0430 .7430

Group 4 (180mg/kg of Extract) .47500* .18708 .021 .0820 .8680

253

Group 5 (5mg/kg of Artesunate) .12500 .18708 .513 -.2680 .5180

Choles

-terol

LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) .19500 .26660 .474 -.3651 .7551

Group 3 (90mg/kg of Extract) .07750 .26660 .775 -.4826 .6376

Group 4 (180mg/kg of Extract) .28500 .26660 .299 -.2751 .8451

Group 5 (5mg/kg of Artesunate) .19250 .26660 .480 -.3676 .7526

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.01250 .26660 .963 -.5476 .5726

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.19500 .26660 .474 -.7551 .3651

Group 3 (90mg/kg of Extract) -.11750 .26660 .665 -.6776 .4426

Group 4 (180mg/kg of Extract) .09000 .26660 .740 -.4701 .6501

Group 5 (5mg/kg of Artesunate) -.00250 .26660 .993 -.5626 .5576

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.18250 .26660 .502 -.7426 .3776

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -.07750 .26660 .775 -.6376 .4826

Group 2 (45 mg/kg Extract) .11750 .26660 .665 -.4426 .6776

Group 4 (180mg/kg of Extract) .20750 .26660 .446 -.3526 .7676

Group 5 (5mg/kg of Artesunate) .11500 .26660 .671 -.4451 .6751

254

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.06500 .26660 .810 -.6251 .4951

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -.28500 .26660 .299 -.8451 .2751

Group 2 (45 mg/kg Extract) -.09000 .26660 .740 -.6501 .4701

Group 3 (90mg/kg of Extract) -.20750 .26660 .446 -.7676 .3526

Group 5 (5mg/kg of Artesunate) -.09250 .26660 .733 -.6526 .4676

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.27250 .26660 .320 -.8326 .2876

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -.19250 .26660 .480 -.7526 .3676

Group 2 (45 mg/kg Extract) .00250 .26660 .993 -.5576 .5626

Group 3 (90mg/kg of Extract) -.11500 .26660 .671 -.6751 .4451

Group 4 (180mg/kg of Extract) .09250 .26660 .733 -.4676 .6526

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.18000 .26660 .508 -.7401 .3801

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -.01250 .26660 .963 -.5726 .5476

Group 2 (45 mg/kg Extract) .18250 .26660 .502 -.3776 .7426

Group 3 (90mg/kg of Extract) .06500 .26660 .810 -.4951 .6251

Group 4 (180mg/kg of Extract) .27250 .26660 .320 -.2876 .8326

255

Group 5 (5mg/kg of Artesunate) .18000 .26660 .508 -.3801 .7401

HDL LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) -.06750 .13062 .612 -.3419 .2069

Group 3 (90mg/kg of Extract) -.04750 .13062 .720 -.3219 .2269

Group 4 (180mg/kg of Extract) -.09750 .13062 .465 -.3719 .1769

Group 5 (5mg/kg of Artesunate) .08500 .13062 .523 -.1894 .3594

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.09250 .13062 .488 -.1819 .3669

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) .06750 .13062 .612 -.2069 .3419

Group 3 (90mg/kg of Extract) .02000 .13062 .880 -.2544 .2944

Group 4 (180mg/kg of Extract) -.03000 .13062 .821 -.3044 .2444

Group 5 (5mg/kg of Artesunate) .15250 .13062 .258 -.1219 .4269

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.16000 .13062 .236 -.1144 .4344

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) .04750 .13062 .720 -.2269 .3219

Group 2 (45 mg/kg Extract) -.02000 .13062 .880 -.2944 .2544

Group 4 (180mg/kg of Extract) -.05000 .13062 .706 -.3244 .2244

Group 5 (5mg/kg of Artesunate) .13250 .13062 .324 -.1419 .4069

256

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.14000 .13062 .298 -.1344 .4144

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) .09750 .13062 .465 -.1769 .3719

Group 2 (45 mg/kg Extract) .03000 .13062 .821 -.2444 .3044

Group 3 (90mg/kg of Extract) .05000 .13062 .706 -.2244 .3244

Group 5 (5mg/kg of Artesunate) .18250 .13062 .179 -.0919 .4569

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.19000 .13062 .163 -.0844 .4644

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -.08500 .13062 .523 -.3594 .1894

Group 2 (45 mg/kg Extract) -.15250 .13062 .258 -.4269 .1219

Group 3 (90mg/kg of Extract) -.13250 .13062 .324 -.4069 .1419

Group 4 (180mg/kg of Extract) -.18250 .13062 .179 -.4569 .0919

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.00750 .13062 .955 -.2669 .2819

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -.09250 .13062 .488 -.3669 .1819

Group 2 (45 mg/kg Extract) -.16000 .13062 .236 -.4344 .1144

Group 3 (90mg/kg of Extract) -.14000 .13062 .298 -.4144 .1344

Group 4 (180mg/kg of Extract) -.19000 .13062 .163 -.4644 .0844

257

Group 5 (5mg/kg of Artesunate) -.00750 .13062 .955 -.2819 .2669

LDL LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) .21000 .34939 .555 -.5240 .9440

Group 3 (90mg/kg of Extract) .09000 .34939 .800 -.6440 .8240

Group 4 (180mg/kg of Extract) .48750 .34939 .180 -.2465 1.2215

Group 5 (5mg/kg of Artesunate) .08000 .34939 .821 -.6540 .8140

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.07500 .34939 .832 -.8090 .6590

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.21000 .34939 .555 -.9440 .5240

Group 3 (90mg/kg of Extract) -.12000 .34939 .735 -.8540 .6140

Group 4 (180mg/kg of Extract) .27750 .34939 .437 -.4565 1.0115

Group 5 (5mg/kg of Artesunate) -.13000 .34939 .714 -.8640 .6040

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.28500 .34939 .425 -1.0190 .4490

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -.09000 .34939 .800 -.8240 .6440

Group 2 (45 mg/kg Extract) .12000 .34939 .735 -.6140 .8540

Group 4 (180mg/kg of Extract) .39750 .34939 .270 -.3365 1.1315

Group 5 (5mg/kg of Artesunate) -.01000 .34939 .977 -.7440 .7240

258

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.16500 .34939 .642 -.8990 .5690

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) -.48750 .34939 .180 -1.2215 .2465

Group 2 (45 mg/kg Extract) -.27750 .34939 .437 -1.0115 .4565

Group 3 (90mg/kg of Extract) -.39750 .34939 .270 -1.1315 .3365

Group 5 (5mg/kg of Artesunate) -.40750 .34939 .259 -1.1415 .3265

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.56250 .34939 .125 -1.2965 .1715

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -.08000 .34939 .821 -.8140 .6540

Group 2 (45 mg/kg Extract) .13000 .34939 .714 -.6040 .8640

Group 3 (90mg/kg of Extract) .01000 .34939 .977 -.7240 .7440

Group 4 (180mg/kg of Extract) .40750 .34939 .259 -.3265 1.1415

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.15500 .34939 .663 -.8890 .5790

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) .07500 .34939 .832 -.6590 .8090

Group 2 (45 mg/kg Extract) .28500 .34939 .425 -.4490 1.0190

Group 3 (90mg/kg of Extract) .16500 .34939 .642 -.5690 .8990

Group 4 (180mg/kg of Extract) .56250 .34939 .125 -.1715 1.2965

259

Group 5 (5mg/kg of Artesunate) .15500 .34939 .663 -.5790 .8890

TAG LSD Group 1 (Positive Control) Group 2 (45 mg/kg Extract) .11750 .10614 .283 -.1055 .3405

Group 3 (90mg/kg of Extract) .08000 .10614 .461 -.1430 .3030

Group 4 (180mg/kg of Extract) -.05250 .10614 .627 -.2755 .1705

Group 5 (5mg/kg of Artesunate) .06750 .10614 .533 -.1555 .2905

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.01250 .10614 .908 -.2355 .2105

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.11750 .10614 .283 -.3405 .1055

Group 3 (90mg/kg of Extract) -.03750 .10614 .728 -.2605 .1855

Group 4 (180mg/kg of Extract) -.17000 .10614 .127 -.3930 .0530

Group 5 (5mg/kg of Artesunate) -.05000 .10614 .643 -.2730 .1730

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.13000 .10614 .236 -.3530 .0930

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -.08000 .10614 .461 -.3030 .1430

Group 2 (45 mg/kg Extract) .03750 .10614 .728 -.1855 .2605

Group 4 (180mg/kg of Extract) -.13250 .10614 .228 -.3555 .0905

Group 5 (5mg/kg of Artesunate) -.01250 .10614 .908 -.2355 .2105

260

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.09250 .10614 .395 -.3155 .1305

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) .05250 .10614 .627 -.1705 .2755

Group 2 (45 mg/kg Extract) .17000 .10614 .127 -.0530 .3930

Group 3 (90mg/kg of Extract) .13250 .10614 .228 -.0905 .3555

Group 5 (5mg/kg of Artesunate) .12000 .10614 .273 -.1030 .3430

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.04000 .10614 .711 -.1830 .2630

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -.06750 .10614 .533 -.2905 .1555

Group 2 (45 mg/kg Extract) .05000 .10614 .643 -.1730 .2730

Group 3 (90mg/kg of Extract) .01250 .10614 .908 -.2105 .2355

Group 4 (180mg/kg of Extract) -.12000 .10614 .273 -.3430 .1030

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.08000 .10614 .461 -.3030 .1430

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) .01250 .10614 .908 -.2105 .2355

Group 2 (45 mg/kg Extract) .13000 .10614 .236 -.0930 .3530

Group 3 (90mg/kg of Extract) .09250 .10614 .395 -.1305 .3155

Group 4 (180mg/kg of Extract) -.04000 .10614 .711 -.2630 .1830

261

Group 5 (5mg/kg of Artesunate) .08000 .10614 .461 -.1430 .3030

*. The mean difference is significant at the 0.05 level.

262

APPENDIX V: Descriptive and Multiple Comparison Tables of Haemalogical

Parameters

Oneway (Group 1: Positive Control)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin 3 Days after innoculation 4 13.5250 .57373 .28687

Day 5 of Treatment 4 11.2750 1.02429 .51214

Day 28 Post-treatment 4 10.5750 .95000 .47500

Total 12 11.7917 1.53295 .44252

Packed Cell Volume 3 Days after innoculation 4 39.7500 1.70783 .85391

Day 5 of Treatment 4 33.7500 2.62996 1.31498

Day 28 Post-treatment 4 30.5000 2.38048 1.19024

Total 12 34.6667 4.49916 1.29880

T_WBC 3 Days after innoculation 4 6.7500 2.06801 1.03401

Day 5 of Treatment 4 11.8000 2.85657 1.42829

Day 28 Post-treatment 4 13.7000 1.90788 .95394

Total 12 10.7500 3.71055 1.07114

RBC 3 Days after innoculation 4 7.8000 2.13229 1.06615

Day 5 of Treatment 4 6.5500 2.37417 1.18708

Day 28 Post-treatment 4 4.5750 .97425 .48713

Total 12 6.3083 2.22688 .64285

Post Hoc Tests

263

Multiple Comparisons

Dependent Variable (I) Days (J) Days Mean Difference (I-J)

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment 2.25000*

Day 28 Post-treatment 2.95000*

Day 5 of Treatment 3 Days after innoculation -2.25000*

Day 28 Post-treatment .70000

Day 28 Post-treatment 3 Days after innoculation -2.95000*

Day 5 of Treatment -.70000

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment 6.00000*

Day 28 Post-treatment 9.25000*

Day 5 of Treatment 3 Days after innoculation -6.00000*

Day 28 Post-treatment 3.25000

Day 28 Post-treatment 3 Days after innoculation -9.25000*

Day 5 of Treatment -3.25000

T_WBC LSD 3 Days after innoculation Day 5 of Treatment -5.05000*

Day 28 Post-treatment -6.95000*

Day 5 of Treatment 3 Days after innoculation 5.05000*

Day 28 Post-treatment -1.90000

Day 28 Post-treatment 3 Days after innoculation 6.95000*

Day 5 of Treatment 1.90000

RBC LSD 3 Days after innoculation Day 5 of Treatment 1.25000

Day 28 Post-treatment 3.22500*

Day 5 of Treatment 3 Days after innoculation -1.25000

Day 28 Post-treatment 1.97500

264

Day 28 Post-treatment 3 Days after innoculation -3.22500*

Day 5 of Treatment -1.97500

*. The mean difference is significant at the 0.05 level.

Multiple Comparisons

Dependent Variable (I) Days (J) Days Sig.

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment .005

Day 28 Post-treatment .001

Day 5 of Treatment 3 Days after innoculation .005

Day 28 Post-treatment .286

Day 28 Post-treatment 3 Days after innoculation .001

Day 5 of Treatment .286

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment .005

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .005

Day 28 Post-treatment .074

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .074

T_WBC LSD 3 Days after innoculation Day 5 of Treatment .013

Day 28 Post-treatment .002

Day 5 of Treatment 3 Days after innoculation .013

Day 28 Post-treatment .276

Day 28 Post-treatment 3 Days after innoculation .002

Day 5 of Treatment .276

265

RBC LSD 3 Days after innoculation Day 5 of Treatment .383

Day 28 Post-treatment .042

Day 5 of Treatment 3 Days after innoculation .383

Day 28 Post-treatment .181

Day 28 Post-treatment 3 Days after innoculation .042

Day 5 of Treatment .181

Multiple Comparisons

Dependent Variable (I) Days (J) Days 95% Confidence Interval

Lower Bound

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment .8553

Day 28 Post-treatment 1.5553

Day 5 of Treatment 3 Days after innoculation -3.6447

Day 28 Post-treatment -.6947

Day 28 Post-treatment 3 Days after innoculation -4.3447

Day 5 of Treatment -2.0947

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment 2.3641

Day 28 Post-treatment 5.6141

Day 5 of Treatment 3 Days after innoculation -9.6359

Day 28 Post-treatment -.3859

Day 28 Post-treatment 3 Days after innoculation -12.8859

Day 5 of Treatment -6.8859

266

T_WBC LSD 3 Days after innoculation Day 5 of Treatment -8.7529

Day 28 Post-treatment -10.6529

Day 5 of Treatment 3 Days after innoculation 1.3471

Day 28 Post-treatment -5.6029

Day 28 Post-treatment 3 Days after innoculation 3.2471

Day 5 of Treatment -1.8029

RBC LSD 3 Days after innoculation Day 5 of Treatment -1.8314

Day 28 Post-treatment .1436

Day 5 of Treatment 3 Days after innoculation -4.3314

Day 28 Post-treatment -1.1064

Day 28 Post-treatment 3 Days after innoculation -6.3064

Day 5 of Treatment -5.0564

Oneway (Group 2: 45mg/kg b.w. of Extract)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin 3 Days after innoculation 4 13.0250 1.40089 .70045

Day 5 of Treatment 4 11.4000 1.20277 .60139

Day 28 Post-treatment 4 10.1750 1.29711 .64856

Total 12 11.5333 1.69563 .48949

Packed Cell Volume 3 Days after innoculation 4 39.2500 3.59398 1.79699

Day 5 of Treatment 4 34.0000 2.94392 1.47196

267

Day 28 Post-treatment 4 30.0000 3.36650 1.68325

Total 12 34.4167 4.96274 1.43262

T_WBC 3 Days after innoculation 4 5.8500 2.69506 1.34753

Day 5 of Treatment 4 8.8250 1.64190 .82095

Day 28 Post-treatment 4 14.7250 3.78539 1.89269

Total 12 9.8000 4.63289 1.33740

RBC 3 Days after innoculation 4 8.1500 1.47535 .73768

Day 5 of Treatment 4 8.5250 1.15289 .57645

Day 28 Post-treatment 4 8.0000 .81650 .40825

Total 12 8.2250 1.09139 .31506

Multiple Comparisons

Dependent Variable (I) Days (J) Days Sig.

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment .112

Day 28 Post-treatment .013

Day 5 of Treatment 3 Days after innoculation .112

Day 28 Post-treatment .216

Day 28 Post-treatment 3 Days after innoculation .013

Day 5 of Treatment .216

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment .052

Day 28 Post-treatment .003

Day 5 of Treatment 3 Days after innoculation .052

Day 28 Post-treatment .122

Day 28 Post-treatment 3 Days after innoculation .003

268

Day 5 of Treatment .122

T_WBC LSD 3 Days after innoculation Day 5 of Treatment .173

Day 28 Post-treatment .002

Day 5 of Treatment 3 Days after innoculation .173

Day 28 Post-treatment .017

Day 28 Post-treatment 3 Days after innoculation .002

Day 5 of Treatment .017

RBC LSD 3 Days after innoculation Day 5 of Treatment .664

Day 28 Post-treatment .861

Day 5 of Treatment 3 Days after innoculation .664

Day 28 Post-treatment .545

Day 28 Post-treatment 3 Days after innoculation .861

Day 5 of Treatment .545

Oneway (Group 3: 90mg/kg b.w. of Extract)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin 3 Days after innoculation 4 13.1000 1.05198 .52599

Day 5 of Treatment 4 11.4750 1.78396 .89198

Day 28 Post-treatment 4 11.2750 1.60702 .80351

Total 12 11.9500 1.61330 .46572

Packed Cell Volume 3 Days after innoculation 4 39.5000 3.10913 1.55456

269

Day 5 of Treatment 4 34.0000 4.08248 2.04124

Day 28 Post-treatment 4 34.0000 4.24264 2.12132

Total 12 35.8333 4.40729 1.27228

T_WBC 3 Days after innoculation 4 5.6500 2.39096 1.19548

Day 5 of Treatment 4 16.1500 2.19317 1.09659

Day 28 Post-treatment 4 16.5000 2.58070 1.29035

Total 12 12.7667 5.68640 1.64152

RBC 3 Days after innoculation 4 7.0500 1.16190 .58095

Day 5 of Treatment 4 8.4750 1.85899 .92949

Day 28 Post-treatment 4 7.8250 1.27115 .63558

Total 12 7.7833 1.45654 .42047

Multiple Comparisons

Dependent Variable (I) Days (J) Days Sig.

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment .163

Day 28 Post-treatment .122

Day 5 of Treatment 3 Days after innoculation .163

Day 28 Post-treatment .856

Day 28 Post-treatment 3 Days after innoculation .122

Day 5 of Treatment .856

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment .074

Day 28 Post-treatment .074

Day 5 of Treatment 3 Days after innoculation .074

270

Day 28 Post-treatment 1.000

Day 28 Post-treatment 3 Days after innoculation .074

Day 5 of Treatment 1.000

T_WBC LSD 3 Days after innoculation Day 5 of Treatment .000

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .000

Day 28 Post-treatment .841

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .841

RBC LSD 3 Days after innoculation Day 5 of Treatment .202

Day 28 Post-treatment .473

Day 5 of Treatment 3 Days after innoculation .202

Day 28 Post-treatment .545

Day 28 Post-treatment 3 Days after innoculation .473

Day 5 of Treatment .545

Oneway (Group 4: 180mg/kg b.w. of Extract)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin 3 Days after innoculation 4 11.0500 1.05040 .52520

Day 5 of Treatment 4 13.8000 .67330 .33665

Day 28 Post-treatment 4 14.2500 .50662 .25331

271

Total 12 13.0333 1.63614 .47231

Packed Cell Volume 3 Days after innoculation 4 33.2500 3.09570 1.54785

Day 5 of Treatment 4 40.0000 1.41421 .70711

Day 28 Post-treatment 4 42.7500 1.70783 .85391

Total 12 38.6667 4.61880 1.33333

T_WBC 3 Days after innoculation 4 3.9250 .86168 .43084

Day 5 of Treatment 4 13.9500 .95743 .47871

Day 28 Post-treatment 4 14.9250 4.20823 2.10411

Total 12 10.9333 5.67856 1.63926

RBC 3 Days after innoculation 4 6.2750 .76322 .38161

Day 5 of Treatment 4 12.3750 1.70171 .85086

Day 28 Post-treatment 4 13.1500 2.15484 1.07742

Total 12 10.6000 3.53939 1.02173

Multiple Comparisons

Dependent Variable (I) Days (J) Days Sig.

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment .001

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .001

Day 28 Post-treatment .434

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .434

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment .002

272

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .002

Day 28 Post-treatment .111

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .111

T_WBC LSD 3 Days after innoculation Day 5 of Treatment .000

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .000

Day 28 Post-treatment .601

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .601

RBC LSD 3 Days after innoculation Day 5 of Treatment .001

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .001

Day 28 Post-treatment .522

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .522

Oneway (Group 5: 5mg/kg b.w. of Artesunate)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin 3 Days after innoculation 4 12.4000 .80000 .40000

273

Day 5 of Treatment 4 13.9500 1.82665 .91333

Day 28 Post-treatment 4 14.2000 .58878 .29439

Total 12 13.5167 1.36770 .39482

Packed Cell Volume 3 Days after innoculation 4 37.5000 2.38048 1.19024

Day 5 of Treatment 4 41.2500 5.67891 2.83945

Day 28 Post-treatment 4 42.0000 1.63299 .81650

Total 12 40.2500 3.91094 1.12899

T_WBC 3 Days after innoculation 4 6.3000 1.96299 .98150

Day 5 of Treatment 4 18.7250 4.49101 2.24550

Day 28 Post-treatment 4 15.3750 1.65202 .82601

Total 12 13.4667 6.11159 1.76426

RBC 3 Days after innoculation 4 6.4500 .33166 .16583

Day 5 of Treatment 4 11.5500 .70475 .35237

Day 28 Post-treatment 4 16.2750 3.49034 1.74517

Total 12 11.4250 4.58776 1.32437

Multiple Comparisons

Dependent Variable (I) Days (J) Days Sig.

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment .101

Day 28 Post-treatment .063

Day 5 of Treatment 3 Days after innoculation .101

Day 28 Post-treatment .775

274

Day 28 Post-treatment 3 Days after innoculation .063

Day 5 of Treatment .775

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment .183

Day 28 Post-treatment .118

Day 5 of Treatment 3 Days after innoculation .183

Day 28 Post-treatment .780

Day 28 Post-treatment 3 Days after innoculation .118

Day 5 of Treatment .780

T_WBC LSD 3 Days after innoculation Day 5 of Treatment .000

Day 28 Post-treatment .002

Day 5 of Treatment 3 Days after innoculation .000

Day 28 Post-treatment .147

Day 28 Post-treatment 3 Days after innoculation .002

Day 5 of Treatment .147

RBC LSD 3 Days after innoculation Day 5 of Treatment .007

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .007

Day 28 Post-treatment .010

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .010

Oneway (Group 6: Negative Control=5mg/kg H20)

275

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin 3 Days after innoculation 4 13.0750 1.88569 .94285

Day 5 of Treatment 4 14.5750 .85000 .42500

Day 28 Post-treatment 4 14.7500 .52599 .26300

Total 12 14.1333 1.36337 .39357

Packed Cell Volume 3 Days after innoculation 4 39.7500 5.67891 2.83945

Day 5 of Treatment 4 43.5000 2.38048 1.19024

Day 28 Post-treatment 4 44.0000 1.63299 .81650

Total 12 42.4167 3.87201 1.11775

T_WBC 3 Days after innoculation 4 6.7000 1.20554 .60277

Day 5 of Treatment 4 11.1000 1.76257 .88129

Day 28 Post-treatment 4 16.6750 2.18384 1.09192

Total 12 11.4917 4.55181 1.31400

RBC 3 Days after innoculation 4 7.3000 .84459 .42230

Day 5 of Treatment 4 11.3250 2.25592 1.12796

Day 28 Post-treatment 4 12.5500 2.49332 1.24666

Total 12 10.3917 2.96048 .85462

Multiple Comparisons

Dependent Variable (I) Days (J) Days Sig.

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment .119

276

Day 28 Post-treatment .087

Day 5 of Treatment 3 Days after innoculation .119

Day 28 Post-treatment .845

Day 28 Post-treatment 3 Days after innoculation .087

Day 5 of Treatment .845

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment .183

Day 28 Post-treatment .137

Day 5 of Treatment 3 Days after innoculation .183

Day 28 Post-treatment .852

Day 28 Post-treatment 3 Days after innoculation .137

Day 5 of Treatment .852

T_WBC LSD 3 Days after innoculation Day 5 of Treatment .006

Day 28 Post-treatment .000

Day 5 of Treatment 3 Days after innoculation .006

Day 28 Post-treatment .002

Day 28 Post-treatment 3 Days after innoculation .000

Day 5 of Treatment .002

RBC LSD 3 Days after innoculation Day 5 of Treatment .019

Day 28 Post-treatment .005

Day 5 of Treatment 3 Days after innoculation .019

Day 28 Post-treatment .409

Day 28 Post-treatment 3 Days after innoculation .005

Day 5 of Treatment .409

277

Multiple Comparisons

Dependent Variable (I) Days (J) Days 95% Confidence Interval

Lower Bound

Haemoglobin LSD 3 Days after innoculation Day 5 of Treatment -3.4710

Day 28 Post-treatment -3.6460

Day 5 of Treatment 3 Days after innoculation -.4710

Day 28 Post-treatment -2.1460

Day 28 Post-treatment 3 Days after innoculation -.2960

Day 5 of Treatment -1.7960

Packed Cell Volume LSD 3 Days after innoculation Day 5 of Treatment -9.6333

Day 28 Post-treatment -10.1333

Day 5 of Treatment 3 Days after innoculation -2.1333

Day 28 Post-treatment -6.3833

Day 28 Post-treatment 3 Days after innoculation -1.6333

Day 5 of Treatment -5.3833

T_WBC LSD 3 Days after innoculation Day 5 of Treatment -7.2208

Day 28 Post-treatment -12.7958

Day 5 of Treatment 3 Days after innoculation 1.5792

Day 28 Post-treatment -8.3958

Day 28 Post-treatment 3 Days after innoculation 7.1542

Day 5 of Treatment 2.7542

278

RBC LSD 3 Days after innoculation Day 5 of Treatment -7.2267

Day 28 Post-treatment -8.4517

Day 5 of Treatment 3 Days after innoculation .8233

Day 28 Post-treatment -4.4267

Day 28 Post-treatment 3 Days after innoculation 2.0483

Day 5 of Treatment -1.9767

Oneway (3 Days After Inoculation)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin Group 1 (Positive Control) 4 13.5250 .57373 .28687

Group 2 (45 mg/kg Extract) 4 13.0250 1.40089 .70045

Group 3 (90mg/kg of Extract) 4 13.1000 1.05198 .52599

Group 4 (180mg/kg of Extract) 4 11.0500 1.05040 .52520

Group 5 (5mg/kg of Artesunate) 4 12.4000 .80000 .40000

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 13.0750 1.88569 .94285

Total 24 12.6958 1.34632 .27482

Packed Cell Volume Group 1 (Positive Control) 4 39.7500 1.70783 .85391

Group 2 (45 mg/kg Extract) 4 39.2500 3.59398 1.79699

Group 3 (90mg/kg of Extract) 4 39.5000 3.10913 1.55456

Group 4 (180mg/kg of Extract) 4 33.2500 3.09570 1.54785

Group 5 (5mg/kg of Artesunate) 4 37.5000 2.38048 1.19024

279

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 39.7500 5.67891 2.83945

Total 24 38.1667 3.89723 .79552

T_WBC Group 1 (Positive Control) 4 6.7500 2.06801 1.03401

Group 2 (45 mg/kg Extract) 4 5.8500 2.69506 1.34753

Group 3 (90mg/kg of Extract) 4 5.6500 2.39096 1.19548

Group 4 (180mg/kg of Extract) 4 3.9250 .86168 .43084

Group 5 (5mg/kg of Artesunate) 4 6.3000 1.96299 .98150

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 6.7000 1.20554 .60277

Total 24 5.8625 1.99822 .40788

RBC Group 1 (Positive Control) 4 7.8000 2.13229 1.06615

Group 2 (45 mg/kg Extract) 4 8.1500 1.47535 .73768

Group 3 (90mg/kg of Extract) 4 7.0500 1.16190 .58095

Group 4 (180mg/kg of Extract) 4 6.2750 .76322 .38161

Group 5 (5mg/kg of Artesunate) 4 6.4500 .33166 .16583

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 7.3000 .84459 .42230

Total 24 7.1708 1.30666 .26672

280

281

Post Hoc Tests

Multiple Comparisons

Dependent Variable (I) Group (J) Group Mean Difference

(I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

Haemoglobin LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) .50000 .85135 .564 -1.2886 2.2886

Group 3 (90mg/kg of Extract) .42500 .85135 .624 -1.3636 2.2136

Group 4 (180mg/kg of Extract) 2.47500* .85135 .009 .6864 4.2636

Group 5 (5mg/kg of Artesunate) 1.12500 .85135 .203 -.6636 2.9136

Group 6 (Negative Control=5mg/kg of Distilled Water) .45000 .85135 .604 -1.3386 2.2386

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.50000 .85135 .564 -2.2886 1.2886

Group 3 (90mg/kg of Extract) -.07500 .85135 .931 -1.8636 1.7136

Group 4 (180mg/kg of Extract) 1.97500* .85135 .032 .1864 3.7636

Group 5 (5mg/kg of Artesunate) .62500 .85135 .472 -1.1636 2.4136

Group 6 (Negative Control=5mg/kg of Distilled Water) -.05000 .85135 .954 -1.8386 1.7386

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -.42500 .85135 .624 -2.2136 1.3636

Group 2 (45 mg/kg Extract) .07500 .85135 .931 -1.7136 1.8636

Group 4 (180mg/kg of Extract) 2.05000* .85135 .027 .2614 3.8386

282

Group 5 (5mg/kg of Artesunate) .70000 .85135 .422 -1.0886 2.4886

Group 6 (Negative Control=5mg/kg of Distilled Water) .02500 .85135 .977 -1.7636 1.8136

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) -2.47500* .85135 .009 -4.2636 -.6864

Group 2 (45 mg/kg Extract) -1.97500* .85135 .032 -3.7636 -.1864

Group 3 (90mg/kg of Extract) -2.05000* .85135 .027 -3.8386 -.2614

Group 5 (5mg/kg of Artesunate) -1.35000 .85135 .130 -3.1386 .4386

Group 6 (Negative Control=5mg/kg of Distilled Water) -2.02500* .85135 .029 -3.8136 -.2364

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -1.12500 .85135 .203 -2.9136 .6636

Group 2 (45 mg/kg Extract) -.62500 .85135 .472 -2.4136 1.1636

Group 3 (90mg/kg of Extract) -.70000 .85135 .422 -2.4886 1.0886

Group 4 (180mg/kg of Extract) 1.35000 .85135 .130 -.4386 3.1386

Group 6 (Negative Control=5mg/kg of Distilled Water) -.67500 .85135 .438 -2.4636 1.1136

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -.45000 .85135 .604 -2.2386 1.3386

Group 2 (45 mg/kg Extract) .05000 .85135 .954 -1.7386 1.8386

Group 3 (90mg/kg of Extract) -.02500 .85135 .977 -1.8136 1.7636

Group 4 (180mg/kg of Extract) 2.02500* .85135 .029 .2364 3.8136

Group 5 (5mg/kg of Artesunate) .67500 .85135 .438 -1.1136 2.4636

283

Packed Cell

Volume

LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) .50000 2.46644 .842 -4.6818 5.6818

Group 3 (90mg/kg of Extract) .25000 2.46644 .920 -4.9318 5.4318

Group 4 (180mg/kg of Extract) 6.50000* 2.46644 .017 1.3182 11.6818

Group 5 (5mg/kg of Artesunate) 2.25000 2.46644 .374 -2.9318 7.4318

Group 6 (Negative Control=5mg/kg of Distilled Water) .00000 2.46644 1.000 -5.1818 5.1818

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.50000 2.46644 .842 -5.6818 4.6818

Group 3 (90mg/kg of Extract) -.25000 2.46644 .920 -5.4318 4.9318

Group 4 (180mg/kg of Extract) 6.00000* 2.46644 .026 .8182 11.1818

Group 5 (5mg/kg of Artesunate) 1.75000 2.46644 .487 -3.4318 6.9318

Group 6 (Negative Control=5mg/kg of Distilled Water) -.50000 2.46644 .842 -5.6818 4.6818

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -.25000 2.46644 .920 -5.4318 4.9318

Group 2 (45 mg/kg Extract) .25000 2.46644 .920 -4.9318 5.4318

Group 4 (180mg/kg of Extract) 6.25000* 2.46644 .021 1.0682 11.4318

Group 5 (5mg/kg of Artesunate) 2.00000 2.46644 .428 -3.1818 7.1818

Group 6 (Negative Control=5mg/kg of Distilled Water) -.25000 2.46644 .920 -5.4318 4.9318

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) -6.50000* 2.46644 .017 -11.6818 -1.3182

Group 2 (45 mg/kg Extract) -6.00000* 2.46644 .026 -11.1818 -.8182

284

Group 3 (90mg/kg of Extract) -6.25000* 2.46644 .021 -11.4318 -1.0682

Group 5 (5mg/kg of Artesunate) -4.25000 2.46644 .102 -9.4318 .9318

Group 6 (Negative Control=5mg/kg of Distilled Water) -6.50000* 2.46644 .017 -11.6818 -1.3182

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -2.25000 2.46644 .374 -7.4318 2.9318

Group 2 (45 mg/kg Extract) -1.75000 2.46644 .487 -6.9318 3.4318

Group 3 (90mg/kg of Extract) -2.00000 2.46644 .428 -7.1818 3.1818

Group 4 (180mg/kg of Extract) 4.25000 2.46644 .102 -.9318 9.4318

Group 6 (Negative Control=5mg/kg of Distilled Water) -2.25000 2.46644 .374 -7.4318 2.9318

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) .00000 2.46644 1.000 -5.1818 5.1818

Group 2 (45 mg/kg Extract) .50000 2.46644 .842 -4.6818 5.6818

Group 3 (90mg/kg of Extract) .25000 2.46644 .920 -4.9318 5.4318

Group 4 (180mg/kg of Extract) 6.50000* 2.46644 .017 1.3182 11.6818

Group 5 (5mg/kg of Artesunate) 2.25000 2.46644 .374 -2.9318 7.4318

T_WBC LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) .90000 1.39361 .527 -2.0279 3.8279

Group 3 (90mg/kg of Extract) 1.10000 1.39361 .440 -1.8279 4.0279

Group 4 (180mg/kg of Extract) 2.82500 1.39361 .058 -.1029 5.7529

Group 5 (5mg/kg of Artesunate) .45000 1.39361 .750 -2.4779 3.3779

285

Group 6 (Negative Control=5mg/kg of Distilled Water) .05000 1.39361 .972 -2.8779 2.9779

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.90000 1.39361 .527 -3.8279 2.0279

Group 3 (90mg/kg of Extract) .20000 1.39361 .887 -2.7279 3.1279

Group 4 (180mg/kg of Extract) 1.92500 1.39361 .184 -1.0029 4.8529

Group 5 (5mg/kg of Artesunate) -.45000 1.39361 .750 -3.3779 2.4779

Group 6 (Negative Control=5mg/kg of Distilled Water) -.85000 1.39361 .550 -3.7779 2.0779

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -1.10000 1.39361 .440 -4.0279 1.8279

Group 2 (45 mg/kg Extract) -.20000 1.39361 .887 -3.1279 2.7279

Group 4 (180mg/kg of Extract) 1.72500 1.39361 .232 -1.2029 4.6529

Group 5 (5mg/kg of Artesunate) -.65000 1.39361 .647 -3.5779 2.2779

Group 6 (Negative Control=5mg/kg of Distilled Water) -1.05000 1.39361 .461 -3.9779 1.8779

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) -2.82500 1.39361 .058 -5.7529 .1029

Group 2 (45 mg/kg Extract) -1.92500 1.39361 .184 -4.8529 1.0029

Group 3 (90mg/kg of Extract) -1.72500 1.39361 .232 -4.6529 1.2029

Group 5 (5mg/kg of Artesunate) -2.37500 1.39361 .106 -5.3029 .5529

Group 6 (Negative Control=5mg/kg of Distilled Water) -2.77500 1.39361 .062 -5.7029 .1529

Group 5 (5mg/kg of Group 1 (Positive Control) -.45000 1.39361 .750 -3.3779 2.4779

286

Artesunate) Group 2 (45 mg/kg Extract) .45000 1.39361 .750 -2.4779 3.3779

Group 3 (90mg/kg of Extract) .65000 1.39361 .647 -2.2779 3.5779

Group 4 (180mg/kg of Extract) 2.37500 1.39361 .106 -.5529 5.3029

Group 6 (Negative Control=5mg/kg of Distilled Water) -.40000 1.39361 .777 -3.3279 2.5279

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -.05000 1.39361 .972 -2.9779 2.8779

Group 2 (45 mg/kg Extract) .85000 1.39361 .550 -2.0779 3.7779

Group 3 (90mg/kg of Extract) 1.05000 1.39361 .461 -1.8779 3.9779

Group 4 (180mg/kg of Extract) 2.77500 1.39361 .062 -.1529 5.7029

Group 5 (5mg/kg of Artesunate) .40000 1.39361 .777 -2.5279 3.3279

RBC LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) -.35000 .88878 .698 -2.2173 1.5173

Group 3 (90mg/kg of Extract) .75000 .88878 .410 -1.1173 2.6173

Group 4 (180mg/kg of Extract) 1.52500 .88878 .103 -.3423 3.3923

Group 5 (5mg/kg of Artesunate) 1.35000 .88878 .146 -.5173 3.2173

Group 6 (Negative Control=5mg/kg of Distilled Water) .50000 .88878 .581 -1.3673 2.3673

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) .35000 .88878 .698 -1.5173 2.2173

Group 3 (90mg/kg of Extract) 1.10000 .88878 .232 -.7673 2.9673

Group 4 (180mg/kg of Extract) 1.87500* .88878 .049 .0077 3.7423

287

Group 5 (5mg/kg of Artesunate) 1.70000 .88878 .072 -.1673 3.5673

Group 6 (Negative Control=5mg/kg of Distilled Water) .85000 .88878 .352 -1.0173 2.7173

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) -.75000 .88878 .410 -2.6173 1.1173

Group 2 (45 mg/kg Extract) -1.10000 .88878 .232 -2.9673 .7673

Group 4 (180mg/kg of Extract) .77500 .88878 .395 -1.0923 2.6423

Group 5 (5mg/kg of Artesunate) .60000 .88878 .508 -1.2673 2.4673

Group 6 (Negative Control=5mg/kg of Distilled Water) -.25000 .88878 .782 -2.1173 1.6173

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) -1.52500 .88878 .103 -3.3923 .3423

Group 2 (45 mg/kg Extract) -1.87500* .88878 .049 -3.7423 -.0077

Group 3 (90mg/kg of Extract) -.77500 .88878 .395 -2.6423 1.0923

Group 5 (5mg/kg of Artesunate) -.17500 .88878 .846 -2.0423 1.6923

Group 6 (Negative Control=5mg/kg of Distilled Water) -1.02500 .88878 .264 -2.8923 .8423

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) -1.35000 .88878 .146 -3.2173 .5173

Group 2 (45 mg/kg Extract) -1.70000 .88878 .072 -3.5673 .1673

Group 3 (90mg/kg of Extract) -.60000 .88878 .508 -2.4673 1.2673

Group 4 (180mg/kg of Extract) .17500 .88878 .846 -1.6923 2.0423

Group 6 (Negative Control=5mg/kg of Distilled Water) -.85000 .88878 .352 -2.7173 1.0173

288

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -.50000 .88878 .581 -2.3673 1.3673

Group 2 (45 mg/kg Extract) -.85000 .88878 .352 -2.7173 1.0173

Group 3 (90mg/kg of Extract) .25000 .88878 .782 -1.6173 2.1173

Group 4 (180mg/kg of Extract) 1.02500 .88878 .264 -.8423 2.8923

Group 5 (5mg/kg of Artesunate) .85000 .88878 .352 -1.0173 2.7173

*. The mean difference is significant at the 0.05 level.

289

Oneway (Day 5 Treatment)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin Group 1 (Positive Control) 4 11.2750 1.02429 .51214

Group 2 (45 mg/kg Extract) 4 11.4000 1.20277 .60139

Group 3 (90mg/kg of Extract) 4 11.4750 1.78396 .89198

Group 4 (180mg/kg of Extract) 4 13.8000 .67330 .33665

Group 5 (5mg/kg of Artesunate) 4 13.9500 1.82665 .91333

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 14.5750 .85000 .42500

Total 24 12.7458 1.82447 .37242

Packed Cell Volume Group 1 (Positive Control) 4 33.7500 2.62996 1.31498

Group 2 (45 mg/kg Extract) 4 34.0000 2.94392 1.47196

Group 3 (90mg/kg of Extract) 4 34.0000 4.08248 2.04124

Group 4 (180mg/kg of Extract) 4 40.0000 1.41421 .70711

Group 5 (5mg/kg of Artesunate) 4 41.2500 5.67891 2.83945

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 43.5000 2.38048 1.19024

Total 24 37.7500 5.08408 1.03778

T_WBC Group 1 (Positive Control) 4 11.8000 2.85657 1.42829

Group 2 (45 mg/kg Extract) 4 8.8250 1.64190 .82095

Group 3 (90mg/kg of Extract) 4 16.1500 2.19317 1.09659

Group 4 (180mg/kg of Extract) 4 13.9500 .95743 .47871

Group 5 (5mg/kg of Artesunate) 4 18.7250 4.49101 2.24550

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 11.1000 1.76257 .88129

Total 24 13.4250 4.06237 .82923

290

RBC Group 1 (Positive Control) 4 6.5500 2.37417 1.18708

Group 2 (45 mg/kg Extract) 4 8.5250 1.15289 .57645

Group 3 (90mg/kg of Extract) 4 8.4750 1.85899 .92949

Group 4 (180mg/kg of Extract) 4 12.3750 1.70171 .85086

Group 5 (5mg/kg of Artesunate) 4 11.5500 .70475 .35237

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 11.3250 2.25592 1.12796

Total 24 9.8000 2.64213 .53932

291

Post Hoc Tests

Multiple Comparisons

Dependent Variable (I) Group (J) Group

Mean

Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

Haemoglobin LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) -.12500 .92154 .894 -2.0611 1.8111

Group 3 (90mg/kg of Extract) -.20000 .92154 .831 -2.1361 1.7361

Group 4 (180mg/kg of Extract) -2.52500* .92154 .013 -4.4611 -.5889

Group 5 (5mg/kg of Artesunate) -2.67500* .92154 .009 -4.6111 -.7389

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-3.30000* .92154 .002 -5.2361 -1.3639

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) .12500 .92154 .894 -1.8111 2.0611

Group 3 (90mg/kg of Extract) -.07500 .92154 .936 -2.0111 1.8611

Group 4 (180mg/kg of Extract) -2.40000* .92154 .018 -4.3361 -.4639

Group 5 (5mg/kg of Artesunate) -2.55000* .92154 .013 -4.4861 -.6139

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-3.17500* .92154 .003 -5.1111 -1.2389

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) .20000 .92154 .831 -1.7361 2.1361

Group 2 (45 mg/kg Extract) .07500 .92154 .936 -1.8611 2.0111

292

Group 4 (180mg/kg of Extract) -2.32500* .92154 .021 -4.2611 -.3889

Group 5 (5mg/kg of Artesunate) -2.47500* .92154 .015 -4.4111 -.5389

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-3.10000* .92154 .003 -5.0361 -1.1639

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) 2.52500* .92154 .013 .5889 4.4611

Group 2 (45 mg/kg Extract) 2.40000* .92154 .018 .4639 4.3361

Group 3 (90mg/kg of Extract) 2.32500* .92154 .021 .3889 4.2611

Group 5 (5mg/kg of Artesunate) -.15000 .92154 .873 -2.0861 1.7861

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.77500 .92154 .411 -2.7111 1.1611

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 2.67500* .92154 .009 .7389 4.6111

Group 2 (45 mg/kg Extract) 2.55000* .92154 .013 .6139 4.4861

Group 3 (90mg/kg of Extract) 2.47500* .92154 .015 .5389 4.4111

Group 4 (180mg/kg of Extract) .15000 .92154 .873 -1.7861 2.0861

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-.62500 .92154 .506 -2.5611 1.3111

Group 6 (Negative

Control=5mg/kg of

Group 1 (Positive Control) 3.30000* .92154 .002 1.3639 5.2361

Group 2 (45 mg/kg Extract) 3.17500* .92154 .003 1.2389 5.1111

293

Distilled Water) Group 3 (90mg/kg of Extract) 3.10000* .92154 .003 1.1639 5.0361

Group 4 (180mg/kg of Extract) .77500 .92154 .411 -1.1611 2.7111

Group 5 (5mg/kg of Artesunate) .62500 .92154 .506 -1.3111 2.5611

Packed Cell

Volume

LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) -.25000 2.45232 .920 -5.4021 4.9021

Group 3 (90mg/kg of Extract) -.25000 2.45232 .920 -5.4021 4.9021

Group 4 (180mg/kg of Extract) -6.25000* 2.45232 .020 -11.4021 -1.0979

Group 5 (5mg/kg of Artesunate) -7.50000* 2.45232 .007 -12.6521 -2.3479

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-9.75000* 2.45232 .001 -14.9021 -4.5979

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) .25000 2.45232 .920 -4.9021 5.4021

Group 3 (90mg/kg of Extract) .00000 2.45232 1.000 -5.1521 5.1521

Group 4 (180mg/kg of Extract) -6.00000* 2.45232 .025 -11.1521 -.8479

Group 5 (5mg/kg of Artesunate) -7.25000* 2.45232 .008 -12.4021 -2.0979

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-9.50000* 2.45232 .001 -14.6521 -4.3479

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) .25000 2.45232 .920 -4.9021 5.4021

Group 2 (45 mg/kg Extract) .00000 2.45232 1.000 -5.1521 5.1521

Group 4 (180mg/kg of Extract) -6.00000* 2.45232 .025 -11.1521 -.8479

294

Group 5 (5mg/kg of Artesunate) -7.25000* 2.45232 .008 -12.4021 -2.0979

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-9.50000* 2.45232 .001 -14.6521 -4.3479

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) 6.25000* 2.45232 .020 1.0979 11.4021

Group 2 (45 mg/kg Extract) 6.00000* 2.45232 .025 .8479 11.1521

Group 3 (90mg/kg of Extract) 6.00000* 2.45232 .025 .8479 11.1521

Group 5 (5mg/kg of Artesunate) -1.25000 2.45232 .616 -6.4021 3.9021

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-3.50000 2.45232 .171 -8.6521 1.6521

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 7.50000* 2.45232 .007 2.3479 12.6521

Group 2 (45 mg/kg Extract) 7.25000* 2.45232 .008 2.0979 12.4021

Group 3 (90mg/kg of Extract) 7.25000* 2.45232 .008 2.0979 12.4021

Group 4 (180mg/kg of Extract) 1.25000 2.45232 .616 -3.9021 6.4021

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-2.25000 2.45232 .371 -7.4021 2.9021

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) 9.75000* 2.45232 .001 4.5979 14.9021

Group 2 (45 mg/kg Extract) 9.50000* 2.45232 .001 4.3479 14.6521

Group 3 (90mg/kg of Extract) 9.50000* 2.45232 .001 4.3479 14.6521

295

Group 4 (180mg/kg of Extract) 3.50000 2.45232 .171 -1.6521 8.6521

Group 5 (5mg/kg of Artesunate) 2.25000 2.45232 .371 -2.9021 7.4021

T_WBC LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) 2.97500 1.82251 .120 -.8539 6.8039

Group 3 (90mg/kg of Extract) -4.35000* 1.82251 .028 -8.1789 -.5211

Group 4 (180mg/kg of Extract) -2.15000 1.82251 .253 -5.9789 1.6789

Group 5 (5mg/kg of Artesunate) -6.92500* 1.82251 .001 -10.7539 -3.0961

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.70000 1.82251 .705 -3.1289 4.5289

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -2.97500 1.82251 .120 -6.8039 .8539

Group 3 (90mg/kg of Extract) -7.32500* 1.82251 .001 -11.1539 -3.4961

Group 4 (180mg/kg of Extract) -5.12500* 1.82251 .012 -8.9539 -1.2961

Group 5 (5mg/kg of Artesunate) -9.90000* 1.82251 .000 -13.7289 -6.0711

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-2.27500 1.82251 .228 -6.1039 1.5539

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) 4.35000* 1.82251 .028 .5211 8.1789

Group 2 (45 mg/kg Extract) 7.32500* 1.82251 .001 3.4961 11.1539

Group 4 (180mg/kg of Extract) 2.20000 1.82251 .243 -1.6289 6.0289

Group 5 (5mg/kg of Artesunate) -2.57500 1.82251 .175 -6.4039 1.2539

296

Group 6 (Negative Control=5mg/kg of

Distilled Water)

5.05000* 1.82251 .013 1.2211 8.8789

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) 2.15000 1.82251 .253 -1.6789 5.9789

Group 2 (45 mg/kg Extract) 5.12500* 1.82251 .012 1.2961 8.9539

Group 3 (90mg/kg of Extract) -2.20000 1.82251 .243 -6.0289 1.6289

Group 5 (5mg/kg of Artesunate) -4.77500* 1.82251 .017 -8.6039 -.9461

Group 6 (Negative Control=5mg/kg of

Distilled Water)

2.85000 1.82251 .135 -.9789 6.6789

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 6.92500* 1.82251 .001 3.0961 10.7539

Group 2 (45 mg/kg Extract) 9.90000* 1.82251 .000 6.0711 13.7289

Group 3 (90mg/kg of Extract) 2.57500 1.82251 .175 -1.2539 6.4039

Group 4 (180mg/kg of Extract) 4.77500* 1.82251 .017 .9461 8.6039

Group 6 (Negative Control=5mg/kg of

Distilled Water)

7.62500* 1.82251 .001 3.7961 11.4539

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) -.70000 1.82251 .705 -4.5289 3.1289

Group 2 (45 mg/kg Extract) 2.27500 1.82251 .228 -1.5539 6.1039

Group 3 (90mg/kg of Extract) -5.05000* 1.82251 .013 -8.8789 -1.2211

Group 4 (180mg/kg of Extract) -2.85000 1.82251 .135 -6.6789 .9789

297

Group 5 (5mg/kg of Artesunate) -7.62500* 1.82251 .001 -11.4539 -3.7961

RBC LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) -1.97500 1.25510 .133 -4.6119 .6619

Group 3 (90mg/kg of Extract) -1.92500 1.25510 .142 -4.5619 .7119

Group 4 (180mg/kg of Extract) -5.82500* 1.25510 .000 -8.4619 -3.1881

Group 5 (5mg/kg of Artesunate) -5.00000* 1.25510 .001 -7.6369 -2.3631

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-4.77500* 1.25510 .001 -7.4119 -2.1381

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) 1.97500 1.25510 .133 -.6619 4.6119

Group 3 (90mg/kg of Extract) .05000 1.25510 .969 -2.5869 2.6869

Group 4 (180mg/kg of Extract) -3.85000* 1.25510 .007 -6.4869 -1.2131

Group 5 (5mg/kg of Artesunate) -3.02500* 1.25510 .027 -5.6619 -.3881

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-2.80000* 1.25510 .039 -5.4369 -.1631

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) 1.92500 1.25510 .142 -.7119 4.5619

Group 2 (45 mg/kg Extract) -.05000 1.25510 .969 -2.6869 2.5869

Group 4 (180mg/kg of Extract) -3.90000* 1.25510 .006 -6.5369 -1.2631

Group 5 (5mg/kg of Artesunate) -3.07500* 1.25510 .025 -5.7119 -.4381

298

Group 6 (Negative Control=5mg/kg of

Distilled Water)

-2.85000* 1.25510 .036 -5.4869 -.2131

Group 4 (180mg/kg of

Extract)

Group 1 (Positive Control) 5.82500* 1.25510 .000 3.1881 8.4619

Group 2 (45 mg/kg Extract) 3.85000* 1.25510 .007 1.2131 6.4869

Group 3 (90mg/kg of Extract) 3.90000* 1.25510 .006 1.2631 6.5369

Group 5 (5mg/kg of Artesunate) .82500 1.25510 .519 -1.8119 3.4619

Group 6 (Negative Control=5mg/kg of

Distilled Water)

1.05000 1.25510 .414 -1.5869 3.6869

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 5.00000* 1.25510 .001 2.3631 7.6369

Group 2 (45 mg/kg Extract) 3.02500* 1.25510 .027 .3881 5.6619

Group 3 (90mg/kg of Extract) 3.07500* 1.25510 .025 .4381 5.7119

Group 4 (180mg/kg of Extract) -.82500 1.25510 .519 -3.4619 1.8119

Group 6 (Negative Control=5mg/kg of

Distilled Water)

.22500 1.25510 .860 -2.4119 2.8619

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) 4.77500* 1.25510 .001 2.1381 7.4119

Group 2 (45 mg/kg Extract) 2.80000* 1.25510 .039 .1631 5.4369

Group 3 (90mg/kg of Extract) 2.85000* 1.25510 .036 .2131 5.4869

Group 4 (180mg/kg of Extract) -1.05000 1.25510 .414 -3.6869 1.5869

299

Group 5 (5mg/kg of Artesunate) -.22500 1.25510 .860 -2.8619 2.4119

*. The mean difference is significant at the 0.05 level.

300

301

Oneway (Day 28 of Post-Treatment)

Descriptives

N Mean Std. Deviation Std. Error

Haemoglobin Group 1 (Positive Control) 4 10.5750 .95000 .47500

Group 2 (45 mg/kg Extract) 4 10.1750 1.29711 .64856

Group 3 (90mg/kg of Extract) 4 11.2750 1.60702 .80351

Group 4 (180mg/kg of Extract) 4 14.2500 .50662 .25331

Group 5 (5mg/kg of Artesunate) 4 14.2000 .58878 .29439

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 14.7500 .52599 .26300

Total 24 12.5375 2.13273 .43534

Packed Cell Volume Group 1 (Positive Control) 4 30.5000 2.38048 1.19024

Group 2 (45 mg/kg Extract) 4 30.0000 3.36650 1.68325

Group 3 (90mg/kg of Extract) 4 34.0000 4.24264 2.12132

Group 4 (180mg/kg of Extract) 4 42.7500 1.70783 .85391

Group 5 (5mg/kg of Artesunate) 4 42.0000 1.63299 .81650

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 44.0000 1.63299 .81650

Total 24 37.2083 6.45371 1.31736

T_WBC Group 1 (Positive Control) 4 13.7000 1.90788 .95394

Group 2 (45 mg/kg Extract) 4 14.7250 3.78539 1.89269

Group 3 (90mg/kg of Extract) 4 16.5000 2.58070 1.29035

Group 4 (180mg/kg of Extract) 4 14.9250 4.20823 2.10411

Group 5 (5mg/kg of Artesunate) 4 15.3750 1.65202 .82601

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 16.6750 2.18384 1.09192

Total 24 15.3167 2.75818 .56301

302

RBC Group 1 (Positive Control) 4 4.5750 .97425 .48713

Group 2 (45 mg/kg Extract) 4 8.0000 .81650 .40825

Group 3 (90mg/kg of Extract) 4 7.8250 1.27115 .63558

Group 4 (180mg/kg of Extract) 4 13.1500 2.15484 1.07742

Group 5 (5mg/kg of Artesunate) 4 16.2750 3.49034 1.74517

Group 6 (Negative Control=5mg/kg of Distilled Water) 4 12.5500 2.49332 1.24666

Total 24 10.3958 4.42773 .90381

303

Post Hoc Tests

Multiple Comparisons

Dependent Variable (I) Group (J) Group

Mean

Difference (I-J)

Std.

Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

Haemoglobin LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) .40000 .70990 .580 -1.0914 1.8914

Group 3 (90mg/kg of Extract) -.70000 .70990 .337 -2.1914 .7914

Group 4 (180mg/kg of Extract) -3.67500* .70990 .000 -5.1664 -2.1836

Group 5 (5mg/kg of Artesunate) -3.62500* .70990 .000 -5.1164 -2.1336

Group 6 (Negative Control=5mg/kg of Distilled Water) -4.17500* .70990 .000 -5.6664 -2.6836

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.40000 .70990 .580 -1.8914 1.0914

Group 3 (90mg/kg of Extract) -1.10000 .70990 .139 -2.5914 .3914

Group 4 (180mg/kg of Extract) -4.07500* .70990 .000 -5.5664 -2.5836

Group 5 (5mg/kg of Artesunate) -4.02500* .70990 .000 -5.5164 -2.5336

Group 6 (Negative Control=5mg/kg of Distilled Water) -4.57500* .70990 .000 -6.0664 -3.0836

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) .70000 .70990 .337 -.7914 2.1914

Group 2 (45 mg/kg Extract) 1.10000 .70990 .139 -.3914 2.5914

Group 4 (180mg/kg of Extract) -2.97500* .70990 .001 -4.4664 -1.4836

Group 5 (5mg/kg of Artesunate) -2.92500* .70990 .001 -4.4164 -1.4336

Group 6 (Negative Control=5mg/kg of Distilled Water) -3.47500* .70990 .000 -4.9664 -1.9836

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) 3.67500* .70990 .000 2.1836 5.1664

Group 2 (45 mg/kg Extract) 4.07500* .70990 .000 2.5836 5.5664

Group 3 (90mg/kg of Extract) 2.97500* .70990 .001 1.4836 4.4664

Group 5 (5mg/kg of Artesunate) .05000 .70990 .945 -1.4414 1.5414

Group 6 (Negative Control=5mg/kg of Distilled Water) -.50000 .70990 .490 -1.9914 .9914

304

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 3.62500* .70990 .000 2.1336 5.1164

Group 2 (45 mg/kg Extract) 4.02500* .70990 .000 2.5336 5.5164

Group 3 (90mg/kg of Extract) 2.92500* .70990 .001 1.4336 4.4164

Group 4 (180mg/kg of Extract) -.05000 .70990 .945 -1.5414 1.4414

Group 6 (Negative Control=5mg/kg of Distilled Water) -.55000 .70990 .449 -2.0414 .9414

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) 4.17500* .70990 .000 2.6836 5.6664

Group 2 (45 mg/kg Extract) 4.57500* .70990 .000 3.0836 6.0664

Group 3 (90mg/kg of Extract) 3.47500* .70990 .000 1.9836 4.9664

Group 4 (180mg/kg of Extract) .50000 .70990 .490 -.9914 1.9914

Group 5 (5mg/kg of Artesunate) .55000 .70990 .449 -.9414 2.0414

Packed Cell

Volume

LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) .50000 1.89846 .795 -3.4885 4.4885

Group 3 (90mg/kg of Extract) -3.50000 1.89846 .082 -7.4885 .4885

Group 4 (180mg/kg of Extract) -12.25000* 1.89846 .000 -16.2385 -8.2615

Group 5 (5mg/kg of Artesunate) -11.50000* 1.89846 .000 -15.4885 -7.5115

Group 6 (Negative Control=5mg/kg of Distilled Water) -13.50000* 1.89846 .000 -17.4885 -9.5115

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) -.50000 1.89846 .795 -4.4885 3.4885

Group 3 (90mg/kg of Extract) -4.00000* 1.89846 .049 -7.9885 -.0115

Group 4 (180mg/kg of Extract) -12.75000* 1.89846 .000 -16.7385 -8.7615

Group 5 (5mg/kg of Artesunate) -12.00000* 1.89846 .000 -15.9885 -8.0115

Group 6 (Negative Control=5mg/kg of Distilled Water) -14.00000* 1.89846 .000 -17.9885 -10.0115

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) 3.50000 1.89846 .082 -.4885 7.4885

Group 2 (45 mg/kg Extract) 4.00000* 1.89846 .049 .0115 7.9885

Group 4 (180mg/kg of Extract) -8.75000* 1.89846 .000 -12.7385 -4.7615

Group 5 (5mg/kg of Artesunate) -8.00000* 1.89846 .001 -11.9885 -4.0115

Group 6 (Negative Control=5mg/kg of Distilled Water) -10.00000* 1.89846 .000 -13.9885 -6.0115

305

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) 12.25000* 1.89846 .000 8.2615 16.2385

Group 2 (45 mg/kg Extract) 12.75000* 1.89846 .000 8.7615 16.7385

Group 3 (90mg/kg of Extract) 8.75000* 1.89846 .000 4.7615 12.7385

Group 5 (5mg/kg of Artesunate) .75000 1.89846 .697 -3.2385 4.7385

Group 6 (Negative Control=5mg/kg of Distilled Water) -1.25000 1.89846 .519 -5.2385 2.7385

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 11.50000* 1.89846 .000 7.5115 15.4885

Group 2 (45 mg/kg Extract) 12.00000* 1.89846 .000 8.0115 15.9885

Group 3 (90mg/kg of Extract) 8.00000* 1.89846 .001 4.0115 11.9885

Group 4 (180mg/kg of Extract) -.75000 1.89846 .697 -4.7385 3.2385

Group 6 (Negative Control=5mg/kg of Distilled Water) -2.00000 1.89846 .306 -5.9885 1.9885

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) 13.50000* 1.89846 .000 9.5115 17.4885

Group 2 (45 mg/kg Extract) 14.00000* 1.89846 .000 10.0115 17.9885

Group 3 (90mg/kg of Extract) 10.00000* 1.89846 .000 6.0115 13.9885

Group 4 (180mg/kg of Extract) 1.25000 1.89846 .519 -2.7385 5.2385

Group 5 (5mg/kg of Artesunate) 2.00000 1.89846 .306 -1.9885 5.9885

T_WBC LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) -1.02500 2.03790 .621 -5.3065 3.2565

Group 3 (90mg/kg of Extract) -2.80000 2.03790 .186 -7.0815 1.4815

Group 4 (180mg/kg of Extract) -1.22500 2.03790 .555 -5.5065 3.0565

Group 5 (5mg/kg of Artesunate) -1.67500 2.03790 .422 -5.9565 2.6065

Group 6 (Negative Control=5mg/kg of Distilled Water) -2.97500 2.03790 .162 -7.2565 1.3065

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) 1.02500 2.03790 .621 -3.2565 5.3065

Group 3 (90mg/kg of Extract) -1.77500 2.03790 .395 -6.0565 2.5065

Group 4 (180mg/kg of Extract) -.20000 2.03790 .923 -4.4815 4.0815

Group 5 (5mg/kg of Artesunate) -.65000 2.03790 .753 -4.9315 3.6315

Group 6 (Negative Control=5mg/kg of Distilled Water) -1.95000 2.03790 .351 -6.2315 2.3315

306

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) 2.80000 2.03790 .186 -1.4815 7.0815

Group 2 (45 mg/kg Extract) 1.77500 2.03790 .395 -2.5065 6.0565

Group 4 (180mg/kg of Extract) 1.57500 2.03790 .450 -2.7065 5.8565

Group 5 (5mg/kg of Artesunate) 1.12500 2.03790 .588 -3.1565 5.4065

Group 6 (Negative Control=5mg/kg of Distilled Water) -.17500 2.03790 .933 -4.4565 4.1065

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) 1.22500 2.03790 .555 -3.0565 5.5065

Group 2 (45 mg/kg Extract) .20000 2.03790 .923 -4.0815 4.4815

Group 3 (90mg/kg of Extract) -1.57500 2.03790 .450 -5.8565 2.7065

Group 5 (5mg/kg of Artesunate) -.45000 2.03790 .828 -4.7315 3.8315

Group 6 (Negative Control=5mg/kg of Distilled Water) -1.75000 2.03790 .402 -6.0315 2.5315

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 1.67500 2.03790 .422 -2.6065 5.9565

Group 2 (45 mg/kg Extract) .65000 2.03790 .753 -3.6315 4.9315

Group 3 (90mg/kg of Extract) -1.12500 2.03790 .588 -5.4065 3.1565

Group 4 (180mg/kg of Extract) .45000 2.03790 .828 -3.8315 4.7315

Group 6 (Negative Control=5mg/kg of Distilled Water) -1.30000 2.03790 .532 -5.5815 2.9815

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) 2.97500 2.03790 .162 -1.3065 7.2565

Group 2 (45 mg/kg Extract) 1.95000 2.03790 .351 -2.3315 6.2315

Group 3 (90mg/kg of Extract) .17500 2.03790 .933 -4.1065 4.4565

Group 4 (180mg/kg of Extract) 1.75000 2.03790 .402 -2.5315 6.0315

Group 5 (5mg/kg of Artesunate) 1.30000 2.03790 .532 -2.9815 5.5815

RBC LSD Group 1 (Positive

Control)

Group 2 (45 mg/kg Extract) -3.42500* 1.47970 .033 -6.5337 -.3163

Group 3 (90mg/kg of Extract) -3.25000* 1.47970 .041 -6.3587 -.1413

Group 4 (180mg/kg of Extract) -8.57500* 1.47970 .000 -11.6837 -5.4663

Group 5 (5mg/kg of Artesunate) -11.70000* 1.47970 .000 -14.8087 -8.5913

Group 6 (Negative Control=5mg/kg of Distilled Water) -7.97500* 1.47970 .000 -11.0837 -4.8663

307

Group 2 (45 mg/kg

Extract)

Group 1 (Positive Control) 3.42500* 1.47970 .033 .3163 6.5337

Group 3 (90mg/kg of Extract) .17500 1.47970 .907 -2.9337 3.2837

Group 4 (180mg/kg of Extract) -5.15000* 1.47970 .003 -8.2587 -2.0413

Group 5 (5mg/kg of Artesunate) -8.27500* 1.47970 .000 -11.3837 -5.1663

Group 6 (Negative Control=5mg/kg of Distilled Water) -4.55000* 1.47970 .007 -7.6587 -1.4413

Group 3 (90mg/kg of

Extract)

Group 1 (Positive Control) 3.25000* 1.47970 .041 .1413 6.3587

Group 2 (45 mg/kg Extract) -.17500 1.47970 .907 -3.2837 2.9337

Group 4 (180mg/kg of Extract) -5.32500* 1.47970 .002 -8.4337 -2.2163

Group 5 (5mg/kg of Artesunate) -8.45000* 1.47970 .000 -11.5587 -5.3413

Group 6 (Negative Control=5mg/kg of Distilled Water) -4.72500* 1.47970 .005 -7.8337 -1.6163

Group 4 (180mg/kg

of Extract)

Group 1 (Positive Control) 8.57500* 1.47970 .000 5.4663 11.6837

Group 2 (45 mg/kg Extract) 5.15000* 1.47970 .003 2.0413 8.2587

Group 3 (90mg/kg of Extract) 5.32500* 1.47970 .002 2.2163 8.4337

Group 5 (5mg/kg of Artesunate) -3.12500* 1.47970 .049 -6.2337 -.0163

Group 6 (Negative Control=5mg/kg of Distilled Water) .60000 1.47970 .690 -2.5087 3.7087

Group 5 (5mg/kg of

Artesunate)

Group 1 (Positive Control) 11.70000* 1.47970 .000 8.5913 14.8087

Group 2 (45 mg/kg Extract) 8.27500* 1.47970 .000 5.1663 11.3837

Group 3 (90mg/kg of Extract) 8.45000* 1.47970 .000 5.3413 11.5587

Group 4 (180mg/kg of Extract) 3.12500* 1.47970 .049 .0163 6.2337

Group 6 (Negative Control=5mg/kg of Distilled Water) 3.72500* 1.47970 .022 .6163 6.8337

Group 6 (Negative

Control=5mg/kg of

Distilled Water)

Group 1 (Positive Control) 7.97500* 1.47970 .000 4.8663 11.0837

Group 2 (45 mg/kg Extract) 4.55000* 1.47970 .007 1.4413 7.6587

Group 3 (90mg/kg of Extract) 4.72500* 1.47970 .005 1.6163 7.8337

Group 4 (180mg/kg of Extract) -.60000 1.47970 .690 -3.7087 2.5087

308

Group 5 (5mg/kg of Artesunate) -3.72500* 1.47970 .022 -6.8337 -.6163

*. The mean difference is significant at the 0.05 level.