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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