i

ii

ONYEMA- AGBOWO EBELE

CYTOTOXICITY AND ANTIRETROVIRAL EFFECTS

OF THE AQUEOUS, METHANOL AND

CHLOROFORM EXTRACTS OF Moringa oleifera

ROOTS.

FACULTY OF BIOLOGICAL SCIENCE

DEPARTMENT OF BIOCHEMISTRY

AVER KELVIN .M

Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

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TITLE

CYTOTOXICITY AND ANTIRETROVIRAL EFFECTS OF THE AQUEOUS,

METHANOL AND CHLOROFORM EXTRACTS OF Moringa oleifera ROOTS.

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR AWARD OF DEGREE OF MASTER OF SCIENCE

(M.Sc) IN PHARMACOLOGICAL BIOCHEMISTRY, UNIVERSITY OF

NIGERIA, NSUKKA.

BY

ONYEMA- AGBOWO EBELE

PG/M.Sc/07/43870

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA NSUKKA

SUPERVISOR: PROF. L.U.S EZEANYIKA

JUNE, 2012

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CERTIFICATION

Onyema-Agbowo Ebele,a postgraduate student of the Department of Biochemistry with the Reg.

No PG/M.Sc/07/43870 has satisfactorily completed her 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.L.U.S.EZEANYIKA PROF.L.U.S.EZEANYIKA

(Supervisor) (Head of Dept)

…………………………………

External Examiner

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DEDICATION

To the glory of God, this work is dedicated to the Holy Spirit, my soul mate, best friend and

mentor. Also my lovely Husband and children, Nissi and Shammah for their love, care and

support throughout the duration of this work.

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ACKNOWLEDGEMENT

My immense gratitude goes to my supervisor, Prof. L.U.S.Ezeanyika. You were not only a

supervisor, you were fatherly. There is no way I can thank you enough for your patience towards my

excesses and work, your dedication was truly remarkable. Your openness, hard work, useful

suggestions and pragmatism shall remain an indelible landmark in my academic life. God bless you

sir.

At various stages of this work, the various contributions of some of my lecturers, Prof Onwurah,

Prof F.C. Chilaka, Prof Alumanah, Prof Uzoegwu,Dr Mrs Chioma Anosike, Dr C.S. Ubani,Dr

Parker Elijah,Dr Enechi, Dr Nwanguma, Dr H.A. Onwubiko,Dr V.N. Ogugua,Dr S.O. Eze, Mr

P.A.C. Egbuna and Mr V.E. Ozougwu were extremely valuable and cannot be forgotten.

A heart-felt thanks goes to Prof O.U. Njoku, who was always willing to give his right arm to

students at any given point in time, you are indeed a blessing sir, thank you so much. Prof

O.F.C.Nwodo, my mentor and my father, you made pharmacology so easy, you are truly an answer

to the prayers of many and a solution to their problems. God bless you and keep you sir. My sister

from another mother, Mrs Ugochi Njoku, thank you so much, it is only the great rewarder that will

pay you for your assistance to me.

As I begin to remember some people whose assistance was significant in this work the list keeps

growing on and on. Worthy of mention is Mr Uchenna Nduka, you were a great inspiration to me

and your counsel was just so wonderful. My wonderful colleagues, Miss Joy Ogana, Mr C.C.

Okonkwo, Mrs Chinelo Nwaorgu, Mrs Florence Nduka (nee Chukwuemeka), Mr Ebubechukwu

Maduekeh, Mr Chinedu Okonkwo, Mr Obinna Ojeeh and Mr Michael Nwankwo. You are a unique

set of people.

This acknowledgement can never be complete without a glowing tribute to my pastor, Pastor Alex

Onuche. Your prayers, encouragement, unwavering support cannot be over quantified, you are a rare

human breed, exactly like Jesus. I am proud to be associated with you. God bless you sir. My

covenant sisters, Mrs Nkechi Ogunjimi, Mrs Chinyere Nnebedum, Mrs Chinwe Onyema, Mrs

Amaka Muomah, Dr Mrs Uju Okoroafor. God bless you my beloved sisters.

My immense gratitude also goes to my God sent husband and children, your support brought me to

this level, may God bless you. The typists, Francisca and Emeka,, my husband’s colleagues, Mrs

Omulu, Oluchi and Onuabuchi who all contributed in typing and photocopying this work. I say a big

thank you and God bless.

I thank almighty God, the ever faithful God who gave me life, strength and the enablement to

accomplish this work. I acknowledged Him ever before I started this work and He has indeed directed

my path towards the successful completion of this work. To Him be all the glory now and forever.

Amen.

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ABSTRACT

One thousand, five hundred grammes (1500g) of Moringa oleifera roots were collected from Prof

Okafor’s plant arboretum in Independence Layout, Enugu, Nigeria and identified by Mr. A.

Ozioko of International Centre for Ethnomedicine and Drug development, Nsukka, Nigeria. The

aqueous, methanol and chloroform extracts were used for phytochemical screening, antinutrient

determination, P24

assay and tissue culture. A total of 54 male albino mice (19.8-39.8g) were used

to determine the median lethal dose using Lorke’s method. Blood samples were collected from

three donors, one HIV negative donor and two HIV positive donors. The blood sample from the

negative volunteer was processed to obtain Peripheral blood mononuclear cells (PBMCs) using a

ficoll hypaque density gradient, centrifuged at 2500 rpm and used for viability testing for

cytotoxicity and the P24 assay for in vitro anti-HIV activity of the extracts. The HIV positive blood

was screened for pre- incubation P24 to determine the P24 antigen concentration and subsequently

used to infect the sero-negative PBMCs, at an infectivity of 5 x 103. Incubation was done at 37

0C

with 5% CO2 for four days inside a phase 3 laminar flow hood. The experimental data were

analyzed using one-way analysis of variance (ANOVA) and Fischer’s least significant difference.

Differences were considered significant at (P < 0.05). Results were presented as mean ± standard

deviation of parameters determined. The methanol and chloroform extracts significantly (P<0.05)

decreased P24 antigen concentration of the PBMCs, from 6.48 pg/ml ± 0.80 to 2.56 pg/ml ±

0.74 and from 6.48 pg/ml ± 0.80 to 3.01 pg/ml ± 0.32 respectively. There was no significant

(P>0.05) decrease in the HIV P24 antigen concentration with the administration of the aqueous

extract (6.48 pg/ml ± 0.80 to 6.43 pg/ml ± 0.95 The methanol extract significantly (P<0.05)

reduced the white blood cells viability from 91% to 61% with an IC50 of 28.5 mg/ml while the

chloroform and the aqueous extracts significantly (P<0.05) reduced the viability of the white cells

from 91% to 76% with an IC50 of 22.3mg/ml and 91% to 15% with an IC50 of 53.3mg/ml

respectively. The phytochemical analysis of the three extracts revealed the presence of bioactive

substances such as reducing sugars, tannins, soluble carbohydrates, glycosides, saponins,

flavonoids, steroids, alkaloids and terpenoids. The antinutrient analysis showed the presence of

phytates, oxalates and haemagluttinins in all the extracts. Trypsin inhibitor was found in minute

quantities in all the extracts. The lethal dose (LD50) of the extracts on mice was found to be less

than or equal to 2900 mg/kg b.w. for the aqueous and methanol extracts and 1000 mg/kg b.w. for

the chloroform extract.

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TABLE OF CONTENTS

Title Page i

Certification ii

Dedication iii

Acknowledgment iv

Abstract v

Table of Contents vi

List of Tables x

List of Figures xi

List of abbreviations xii

Chapter one: Introduction

1.1 Moringa oleifera 3

1.2 Geographical distribution 3

1.3 Nutritional benefits 7

1.4 Other economic uses 16

1.5 Moringa oleifera roots in traditional medicine 17

1.6 Scientific evidence for some of the uses of Moringa oleifera roots in Traditional medicine

17

1.7 Phytochemistry 18

1.8 Moringa oleifera roots and HIV/AIDS 21

1.8.1 History of HIV/AIDS 21

1.8.2 Epidemiology of HIV/AIDS 21

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1.8.3 Impact of HIV/AIDS 22

1.8.4 Stages and symptoms of HIV Disease 24

1.8.5 Structure and Genome of HIV 25

1.8.5.1 Structural proteins 27

1.8.5.2 Regulatory proteins 28

1.8.5.3 Accessory proteins 29

1.8.5.4 Molecular heterogeneity of HIV 30

1.8.5.5 Viral enzymes 31

1.8.6 HIV co-receptors 35

1.8.7 HIV in children 35

1.8.8 Viral dynamics of HIV 35

1.8.9 CD4 T- cells 36

1.8.10 Immune responses against HIV 36

1.8.10.1 Mechanism of immune responses against HIV 37

1.8.11 Laboratory diagnosis 38

1.8.12 HIV Vaccines 40

1.9 Anti-retroviral drug therapy 40

1.9.1 Highly active anti-retroviral therapy (HAART) 42

1.9.2 Herbal remedies and HIV/AIDS therapy 43

1.10 Rationale of the study 45

1.11 Aims 46

1.12 Research Specific objectives 46

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CHAPTER TWO (MATERIALS AND METHODS)

2.1 Animals 47

2.1.1 Human studies 47

2.1.2 Plant materials 47

2.1.3 Chemicals and reagents 47

2.1.4 Equipment/instruments 48

2.2 Experimental design 48

2.3 Method of preparing tissue culture media 51

2.3.1 RPMI culture media preparation 51

2.3.2 Determination of antinutrients 51

2.3.2.1 Determination of phytates 51

2.3.2.2 Determination of oxalates 52

2.3.2.3 Trypsin inhibitors 52

2.3.2.4 Determination of phytohaemaglutinins 54

2.4 Phytochemical studies on Moringa oleifera root extracts (qualitative) 54

2.4.1 Test for carbohydrates 55

2.4.2 Test for alkaloids 55

2.4.3 Test for reducing sugars 56

2.4.4 Test for glycosides 56

2.4.5 Test for saponins 56

2.4.6 Test for tannins 57

2.4.7 Test for flavonoids 57

2.4.8 Test for fats and oils 58

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2.4.9 Test for steroids and terpenoids 58

2.4.10 Test for acidic compounds 58

2.4.11 Test for resins 59

2.5 Quantitative analysis of the aqueous ,methanol and chloroform extracts of Moringa oleifera

roots 59

2.5.1 Flavonoids 59

2.5.2 Reducing sugars 59

2.5.3 Glycosides 59

2.5.4 Tannins 60

2.5.5 Cyanides 60

2.5.6 Soluble carbohydrates 60

2.5.7 Steroids 60

2.5.8 Saponins 60

2.5.9 Alkaloids 61

2.6 Harvesting of PBMCs from blood 61

2.6.1 White cell count and viability testing 62

2.7 Determination of IC50 of plant extracts 63

2.8 Determination of P24 antigen using 4th Generation HIV P24 Elisa kit. 64

2.9 Statistical analysis 69

CHAPTER THREE: RESULTS

3.1 Extract yield 70

3.2 Results of toxicity testing 70

3.3 Phytochemical composition 70

3.4 Results of antinutrient determination 72

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3.5 Results of IC50 of extracts 73

3.6 Results of P24 Assay concentrations 74

3.7 Effects of the extracts on viability (cytotoxicity) 75

3.7.1 Calculations and interpretation of results 75

3.8 Results of viability of the PBMCs 76

CHAPTER FOUR: DISCUSSION

4.1 Discussions 77

4.2 Conclusion 83

4.3 Indications for further research 83

REFERENCES 85

APPENDICES 95

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LIST OF TABLES

Table 1: Nutritional values of Moringa oleifera pods and fresh and dried leaves 8

Table 2: Moringa oleifera leaves compared to common leaves and fruits 10

Table 3: Comparison of the nutritional values of M .oleifera with those of other sources 11

Table 4: Recommended daily allowances for a child (1-3 years old) and nursing mother 12

Table 5: RDA for children 1-3 years 13

Table 6: RDA for lactating mothers 14

Table 7: Qualititative analysis of phytochemicals 71

Table 8: Quantitative analysis of phytochemicals 72

Table 9: Table showing antinutrient composition of M .oleifera roots 73

Table 10: Results of IC50 of the extracts used in the stimulation experiment 73

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LIST OF FIGURES

Figure 1: Photograph of Moringa oleifera tree with leaves and stem 5

Figure 2: The leaves and flowers of Moringa oleifera tree 6

Figure 3: The roots of Moringa oleifera tree 7

Figure 4: Structure of alkaloid moringine 19

Figure 5: Structures of some isolated and characterized phytochemicals from Moringa

oleifera plant. 20

Figure 6: The HIV genome 26

Figure 7: HIV long terminal repeat 29

Figure 8: Life cycle of HIV 33

Figure 9: Graph showing the P24 concentrations before and after addition of extracts. 74

Figure 10: Graph showing the viability of the Peripheral blood mononuclear cells

(PBMCs) before and after exposure to extracts. 76

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LIST OF ABBREVIATIONS

AB - Antibody

ADCC - Antibody dependent cell mediated cytotoxicity

AIDS - Acquired Immunodeficiency Syndrome

ANOVA - Analysis of variance

BBB - Blood brain barrier

CCR5 - Chemokine receptor

CDC - Center for Disease Control and Prevention

CTLs - CD8 Cytotoxic Killer Lymphocytes

CXCR4 - C-X-C Chemokine receptor type 4

CSF - Cerebrospinal fluid

DMSO - Dimethyl sulphoxide

DNA - Deoxyribonucleic acid

ECF - Endocervical fluid

ELISA - Enzyme – linked Immunosorbent assay

Gag gene - Group Specific antigen

gp - Glycoprotein

HAART - Highly Active Anti-retroviral Therapy

HIV - Human Immunodeficiency Virus

HRP - Horse Radish Peroxidase

IC50 - Inhibitory Concentration

IL – Interleukin-1

LD50 - Lethal Dose

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LTNPs - Long Term Non – Progressors

LTR - Long terminal Repeats

MHC - Major Histocompatibility complex

MTT - (3-(4, 5- dimethyl thiazol -2-yl) -2, 5-diphenyl tetrasodium bromide)

Nef gene - Negative factor gene

NK - Natural Killer cells

NNRTI’S - Non nucleoside reverse transcriptase inhibitors

NRTI’S - Nucleotide/Nucleoside Reverse transcriptase inhibitors

OD - Optical Density

PBMC - Peripheral Blood mononuclear cells

PCR - Polymerase chain reaction

RCLB - Red cell lysis buffer

RDA - Recommended Daily Allowance

RNA - Ribonucleic acid

UNAIDS - United Nations Joint Action against AIDS

USAID - United States Aid for International Development

WHO - World Health Organization

xvii

CYTOTOXICITY AND ANTIRETROVIRAL EFFECTS OF THE

AQUEOUS, METHANOL AND CHLOROFORM EXTRACTS

OF Moringa oleifera ROOTS.

BY

ONYEMA- AGBOWO EBELE

PG/M.Sc/07/43870

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA NSUKKA

JUNE, 2012

xviii