1

Digitally Signed by: Content manager’s Name

DN : CN = Weabmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

Nwamarah Uche

Faculty of BIOLOGYCAL SCIENSE

DEPARTMENT OF BIOCHEMISTRY

A comparative study of the effects of aqueous leaf extracts of moringa oleifera and telfairia occidentalis on some

biochemical and haematological parameters in wistar rats

AGHARA, IFEYINWA DOROTHY

(PG/ M.Sc/10/57053)

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A COMPARATIVE STUDY OF THE EFFECTS OF

AQUEOUS LEAF EXTRACTS OF Moringa oleifera AND

Telfairia occidentalis ON SOME BIOCHEMICAL AND

HAEMATOLOGICAL PARAMETERS IN WISTAR RATS

BY

AGHARA, IFEYINWA DOROTHY

(PG/ M.Sc/10/57053)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

FEBRUARY, 2014

3

TITLE PAGE

A COMPARATIVE STUDY OF THE EFFECTS OF AQUEOUS LEAF EXTRACTS OF Moringa oleifera AND Telfairia occidentalis ON SOME

BIOCHEMICAL AND HAEMATOLOGICAL PARAMETERS IN WISTAR RATS

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIRMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF

SCIENCE (M.Sc) IN BIOCHEMISTRY (NUTRITIONAL BIOCHEMISTRY), UNIVERSITY OF NIGERIA, NSUKKA

BY

AGHARA, IFEYINWA DOROTHY (PG/M.Sc/10/57053)

DEPARTMENT OF BICHEMISTRY UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISOR: PROF. L.U.S EZEANYIKA

FEBRUARY, 2014

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CERTIFICATION

AGHARA, Ifeyinwa Dorothy, a post graduate student with Registration Number

PG/M.Sc/10/57053 in the Department of Biochemistry has satisfactorily completed the

requirements for the course work and research work for the award of the degree of Master of

Science (M.Sc) in Biochemistry (Nutritional Biochemistry). The work embodied in this

report is original and has not been submitted in part or full for any diploma or degree of this

or any other University.

............................................ .................................................

PROF. L.U.S EZEANYIKA PROF. OFC NWODO

(Supervisor) (Head of Department)

.............................................................

EXTERNAL EXAMINER

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DEDICATION

This work is dedicated to the Almighty God for His providence, mercies and grace and to my

parents Mr. Michael and Mrs. Jacinta Aghara for their doggedness and sacrifices.

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ACKNOWLEGDEMENT

I express my gratitude to Almighty God for seeing this work to a conclusive end. The success

of this research could not have been achieved without the contributions of numerous people. I

wish to thank my supervisor Prof. L.U.S. Ezeanyika for his tolerance, patience and

instructions. Amidst your tight schedule, you still spared time for my work. I say a big thank

you.

My gratitude goes first to the Head, Department of Biochemistry, Prof. OFC Nwodo for his

contribution and advice

I am also deeply indebted to my lecturers Prof. O.U. Njoku, Prof. P.N. Uzoegwu, Prof. F.C.

Chilaka, Prof. I.N.E. Onwurah, Prof. E.O. Alumanah, Prof. O. Obidoa, Dr. C.S. Ubani, Dr.

(Mrs) C.A. Anosike, Dr. P. E Joshua, Dr. S.O.O Eze, Dr. V.N. Ogugua, Dr. O.C. Enechi, Dr.

H.A. Onwubiko, Mr. P.A.C Egbuna, Mr. O.E. Ikwuagwu, Mr.V.E.O. Ozougwu, Mrs U.O

Njoku, Mrs M.N. Awachie, Mrs. C.I. Ezekwe and a host of others, for the knowledge and

wisdom they imparted into me.

I am sincerely indebted to the sacrifices of my parents Mr. Michael and Mrs. Jacinta Aghara.

Thank you for your perseverance, spiritual, moral and financial support in the course of this

work. To my siblings: Kingsley, Perpetual, Ernest, Peter, Justina, Lucy and Victoria, you

people “add colour to my world”. Thank you all for your prayers and support. I Love you all.

I want to thank all post graduate students of the Department of Biochemistry, University of

Nigeria, Nsukka, for their encouragement and constructive criticisms in the course of this

research. My friends: Okonkwo, C. C., Attamah, J.C., Okey E.N., Osuji, A.C., Okoye, A.C.

and Umeani, I. Thank you all for your support. I want to also thank Ugwuoke, C. you were an

instrument in God’s hands. Thank you very much. God bless you.

I want to also thank all those who in one way or the other contributed to the success of this

work but because of want of space could not be mentioned. God bless you all.

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ABSTRACT

This work was done to ascertain the rationale for the use of the leaves of two vegetables Moringa oleifera and Telfairia occidentalis as traditional haematinics and as such compare their blood boosting capacity with their polyherbal formulation. The aqueous extracts of the leaves and their combined extract were tested for haematinic effects in weanling albino rats. Blood parameters such as Red Blood Cell (RBC) count, White Blood Cell (WBC) count, Haemoglobin concentration (Hb) and Haematocrit were measured. Analyses of the vitamin and mineral contents of the aqueous extracts of the leaves were carried out while phytochemical analyses of the extracts as well as the LD50 and their effect on the hepatocytes were also determined using standard methods. The oral LD50 value of the aqueous extracts was greater than 5000 mg/kg, indicating the high safety profile of Moringa oleifera and

Telfairia occidentalis. Vitamin and mineral analysis showed that Telfairia occidentalis had a higher amount of vitamin B6 (0.88±0.35mg/g) and iron (1.38±0.18 mg/g) than Moringa

oleifera (0.84±.00 mg/g) and (0.70±0.58 mg/g) respectively. However, Moringa oleifera had higher folic acid content (0.37±0.06 mg/g) than Telfairia occidentalis (0.26±0.01 mg/g). Phytochemical analysis of the extracts indicated the presence of alkaloids, flavonoids, terpenoids, carbohydrates, protein and saponins in both extracts while resins and reducing sugar were absent in both extracts. However, cyanogenic glycosides were present only in Moringa oleifera extract. Quantitative phytochemical analysis showed that Moringa oleifera

contains a higher amount of alkaloids and tannins than Telfairia occidentalis. The alkaloid contents of Moringa oleifera and Telfairia occidentalis were 21.53 ± 0.53 mg/g and 12.15 ± 0.42 mg/g respectively and the tannin contents were 7.79 ± 0.05mg/g and 5.58 ± 0.02 mg/g respectively. However, the flavonoid and sterol content of Telfairia occidentalis were slightly higher than the amount in Moringa oleifera. The flavonoid content of Telfairia occidentalis and Moringa oleifera were 18.50 ± 0.14 mg/g and15.42 ± 0.10 mg/g respectively while the sterol contents were 19.92 ±0.55 mg/g and 15.28±0.16 mg/g. The results of the effect of the extracts on the haematopoietic system indicate that oral administration of 20 ml/kg body weight of the aqueous extract of Telfairia occidentalis and 40ml/kg body weight of Moringa

oleifera extract/day exhibited a significant (P < 0.05) increase in haematinic activity by increasing the blood parameters viz, Hb, PCV and RBC. Results from the study showed that Telfairia occidentalis (20ml/kg body weight of the extract) had higher haematinic potency than Moringa oleifera even at 40ml/kg body weight without posing a threat to the hepatocytes but when combined, the extracts decreased the Hb, RBC and PCV. The findings reveal a high content of haematopoetic nutrients in leaves of Telfairia occidentalis and Moringa oleifera

thus justifying their use in the management of anaemia. However, the use of the polyherbal formulation of the aqueous extracts of these leaves showed an antagonistic effect on the haematological indices viz Hb, PCV, RBC and platelet count, and also on the liver by increasing the activity of ALP in the serum.

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TABLE OF CONTENTS

PAGE

Title Page … … … … … … … .;.. … … ii

Certification … … … … … … … … … … iii

Dedication … … … …. … … … … … … iv

Acknowledgement … … … … … … … … … v

Abstract … … … … … … … … … vi

Table of Contents … … … … … … … … … vii

List of Tables … … … … … … … … … xii

List of Plates … … … … … … … … … … xiii

List of Figures … … … … … … … … … xiv

List of Abbreviations … … … … ... … … … … xv

CHAPTER ONE: INTRODUCTION

1.1 Background of the study ... ... ... ... ... ... ... 1

1.2 Moringa oleifera ... ... ... ... ... ... ... .... 2

1.2.1 Scientific classification of Moringa oleifera ... ... ... ... ... 3

1.2.2 Medicinal, industrial and nutritional importance of Moringa oleifera ... 4

1.2.2.1 Medicinal uses of Moringa oleifera ... ... ... ... ... ... 4

1.2.2.2 Industrial uses of Moringa oleifera … … … … … … 5

1.2.2.3 Nutritional uses of Moringa oleifera … … … … … … 5

1.3 Telfairia occidentalis … … … … … … … … 6

1.3.1 Scientific classification of Telfairia occidentalis … … … … 7

1.3.2 Medicinal, industrial and nutritional importance of Telfairia occidentalis … 8

9

1.3.2.1 Medicinal uses of Telfairia occidentalis … … … … … 8

1.3.2.2 Industrial uses of Telfairia occidentalis … … … … … 9

1.3.2.3 Nutritional uses of Telfairia occidentalis … … … … … 9

1.4 Phytochemicals … … … … … … … … 10

1.4.1 Phytochemical constituents of plants ... ... ... ... ... .... 10

1.4.1.1 Terpenoids … … … … … … … … … 10

1.4.1.2 Flavonoids … … … … … … … … … 11

1.4.1.3 Saponins … … … … … … … … … 11

1.4.1.4 Tannins … … … … … … … … … 12

1.4.1.5 Steroids … … … … … … … … … 13

1.4.1.6 Alkaloids … … … … … … … … … 13

1.5 Haematopoiesis ... … … … … … … … 14

1.5.1 Erythropoietin … … … … … … … … 14

1.5.2 Vitamin B12 … … … … … … … … … 15

1.5.3 Folic acid … … … … … … … … … 15

1.5.4 Iron … … … … … … … … … … 16

1.6 Haematological indices … … … … … … … 16

1.6.1 Red blood cell count (erythrocytes) … … … … … 17

1.6.2 Haemoglobin (Hb) … … … … … … … … 17

1.6.3 Packed cell volume (PCV) … … … … … … … 17

1.6.4 Platelet count … … … … … … … … … 17

1.6.5 White blood cell count (WBCs) … … … … … … 18

1.7 The liver … … … … … … … … … 19

1.7.1 Serum enzymes marker of involved in hepatic disorder … … … 19

1.8 Aim of study … … … … … … … … … 20

1.9 Objectives of the research … … … … … … … 20

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials … … … … … … … … … 21

2.1.1 Plant Materials … … … … … … … … 21

2.1.2 Animals … … … … … … … … … 21

2.1.3 Equipments … … … … … … … … … 21

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2.1.4 Chemicals and Reagents … … … … … … … 22

2.2 Methodology … … … … … … … … … 23

2.2.1 Preparation of plant material … … … … … …. … 23

2.2.2 Preparation of reagents for phytochemical analysis … … … … 23

2.3 Qualitative phytochemical analysis of Moringa oleifera and Telfairia occidentalis leaves ... ... ... ... ... ... ... 24

2.3.1 Test for alkaloids … … … … … … … … 24

2.3.2 Test for flavonoids … … … … … … … … 24

2.3.3 Test for glycosides … … … … … … … … 24

2.3.4 Test for proteins … … … … … … … … 25

2.3.5 Test for carbohydrates… … … … … … … … 25

2.3.6 Test for reducing sugars … … … … … … … 25

2.3.7 Test for saponins … … … … … … … … 25

2.3.8 Test for tannins … … … … … … … … 26

2.3.9 Test for oils … … … … … … … … … 26

2.3.10 Test for resins … … … … … … … … … 26

2.3.11 Test for terpenoids and steroids … … … … … … 26

2.4 Quantitative phytochemical analysis of Moringa oleifera and Telfairia occidentalis leaves ... ... ... ... ... ... ... 27

2.4.1 Determination of Alkaloid … … … … … … … 27

2.4.2 Determination of Flavonoid … … … … … … … 27

2.4.3 Determination of Steroids ... ... ... ... ... ... ... 27

2.5 Determination of antinutrient contents of Moringa oleifera and Telfairia occidentalis leaves ... ... ... ... ... ... ... 27

2.5.1 Determination of Tannins ... ... ... ... ... ... ... 27

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2.5.2 Determination of Cyanogenic glycoside ... ... ... ... ... 28 2.6 Acute toxicity (LD50) of the aqueous leaf extracts of Moringa oleifera and

Telfairia occidentalis ... ... ...... ... ... ... ... ... 28

2.7 Determination of vitamin and mineral contents of the aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis ... ... ... 28

2.7.1 Vitamin B6 ... ... ... ... .... ... ... ... ... 28 2.7.2 Folic acid ... ... ... ... ... ... ... ... ... 29 2.7.3 Iron ... ... ... ... ... ... ... ... ... ... 29 2.8 Experimental design for the haematological studies … … … … 29

2.9 Haematological parameters of rats treated with extracts of Telfairia occidentalis

and Moringa oleifera leaves ... ... ... ... ... ... 29

2.9.1 Determination of Packed cell volume … … … … … … 30

2.9.2 Determination of haemoglobin (Hb) concentration … … … … 30

2.9.3 Determination of Red Blood Cell (RBC) counts … … … … 31

2.9.4 Determination of White Blood Cell (WBC) counts … … … … 31

2.9.5 Determination of differential White Blood Cell counts ... ... ... 32

2.10 Liver function test of rats treated with aqueous extracts of Telfairia occidentalis and Moringa oleifera leaves ... ... ... 33

2.10.1 Determination of Alanine Aminotransferase (ALT) … … … … 33

2.10.2 Determination of Aspartate Aminotransferase (AST) … … … 33

2.10.3 Determination of alkaline phosphatase (ALP) … … … … 34

2.11 Statistical analysis … … … … … … … … 34

CHAPTER THREE: RESULTS

3.1 Phytochemical analysis of the leaves of Telfairia occidentalis and

Moringa oleifera … … … … … … … … 35

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3.2 Quantitative phytochemical constituents of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis … … … … … 36

3.3 Acute toxicity (LD50) of the aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis ... ... ... ... ... 37

3.4 Vitamin and mineral contents of Telfairia occidentalis and

Moringa oleifera leaves ... ... ... ... ... ... ... 38 3.5 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Haemoglobin concentration (Hb conc.) ... ... 39

3.6 Effect of the aqueous leaf extracts of Moringa oleifera Telfairia occidentalis on the Packed Cell Volume ... ... ... ... 40 3.7 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Red Blood Cells ... ... ... ... ... 41 3.8 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Platelet count ... ... ... ... ... 42

3.9 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the White Blood Cells … … ... ... 43

3.10 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Neutrophil counts ... ... ... ... 44 3.11 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Lymphocyte counts ... ... ... ... 45 3.12 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Aspartate Transaminase (AST) ... ... ... 46 3.13 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Alanine Transaminase (ALT) … … … 47

3.14 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Alkaline Phosphatase (ALP) … … … 48

CHAPTER FOUR: DISCUSSION

4.1 Discussion … … … … … … … … … 49

4.2 Conclusion … … … … … … … … … 54

4.3 Suggestion for further studies ... ... ... ... ... ... 55

REFERENCES … … … … … … … … … 56

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Appendices ... ... ... ... ... ... ... ... ... ... 68

LIST OF TABLES

Table 1: Normal ranges of the different blood cells ... ... ... ... ... 19

Table 2: Absorbance and activity of ALT in the serum ... ... ... ... 68

Table 3: Absorbance and activity of AST in the serum ... ... ... ... 68

Table 4: Phytochemical analysis of the leaves of Moringa oleifera and Telfairia occidentalis ... ... ... ... ... ... ... 34 Table 5: Quantitative phytochemical contents of the aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis ... ... ... ... 35 Table 6: Phase I of the acute toxicity (LD50) test of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis ... ... ... ... 69

Table 7: Phase II of the acute toxicity (LD50) test of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis ... ... ... ... 69

Table 8: Vitamin and mineral constituents of aqueous extracts of Telfairia occidentalis and Moringa oleifera leaves ... ... ... ... 37

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LIST OF PLATES

Plate 1: The leaves of Moringa oleifera ... ... ... ... ... ... 4

Plate 2: The pods of Moringa oleifera ... ... ... ... ... ... 4

Plate 3: The leaves and pods of Telfairia occidentalis and

Moringa oleifera ... ... ... ... ... ... .... ... 7

LIST OF FIGURES

Fig.1 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Haemoglobin concentration (Hb conc.) ... 39 Fig.2 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Packed Cell Volume (PCV) ... ... ... 40

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Fig.3 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Red Blood Cells (RBCs) ... ... ... 41

Fig.4 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Platelet count ... ... ... ... ... 42

Fig.5 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the White Blood Cells (WBCs) ... ... ... 43

Fig.6 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Neutrophil counts ... ... ... ... 44 Fig.7 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Lymphocyte counts ... ... ... ... 45 Fig.8 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Aspartate Transaminase (AST) ... ... 46 Fig.9 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Alanine Transaminase (ALT) ... ... ... 47

Fig.10 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the Alkaline Phosphatase (ALP) ... ... ... 48

LIST OF ABBREVIATIONS

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

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ANOVA Analysis of variance

AST Aspartate Aminotransferase

B.w Body weight

DNA Deoxyribonucleic acid

EDTA Ethylene Diammine Tetraacetic Acid

EPO Erythropoietin

Hb Haemoglobin

HSCs Haematopoietic Stem Cells

LD Lethal Dose

MOLM Moringa oleifera Leaf Meal

PCV Packed Cell Volume

RBC Red Blood Cell

SPSS Statistical package for social sciences

THF 5, 10- methylene Tetrahydrofolate

WBC White Blood Cell

WHO World Health Organization

CHAPTER ONE

INTRODUCTION

1.1 Background of the study

The practice of traditional medicine is as old as the origin of man (Doughari et al., 2009).

The use of plants in traditional medicine referred to as herbalism or botanical medicine

(Evans, 2002) falls outside the mainstream of the Western or Orthodox medicine. It has been

estimated that about two third of the world’s population (mainly in the developing countries)

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rely on traditional medicine as their primary form of health care (Sumner, 2000). The use of

traditional medicine in the treatment and management of diseases in the African continent

cannot fade away and this could be attributed to the socio-cultural, socio-economic, lack of

basic health care and qualified personnel (Elujoba et al., 2005). Plants contain active

components such as anthraquinones, flavonoids, glycosides, saponins, tannins, etc., which

possess medicinal properties that are harnessed for the treatment of different diseases

(Chevalier, 2000). The active ingredients for a vast number of pharmaceutically derived

medications contain components originating from phytochemicals. These active substances

that contain the healing property are known as the active principles and are found to differ

from plant to plant (Chevalier, 2000). Among these plants are the vegetables whose part(s) are

eaten as supporting food or main dishes and which could be aromatic, bitter or tasteless

(Edema, 1987).

Vegetables vary considerably in their nutrient contents and are good sources of vitamins,

essential amino acids, proteins, as well as minerals and antioxidants (Fasuyi, 2006). They are

included in meals mainly for their nutritional value although some are reserved for the sick

due to their medicinal properties (Okafor, 1983). Generally, the active principles found in

vegetables can be extracted and used in different forms which include infusions, syrups,

concoctions, decoctions, infusion oils, essential oils, ointments and creams in the

treatment/management and prevention of some diseases (Sofowora, 1996).

The human body is known to produce billions of new red blood cells, and other blood

components which replace blood cells that are lost due to normal cell turnover processes,

illness or trauma (Akashi et al., 1999). All the mature blood cells in the body are generated

from a relatively small number of haematopoietic stem cells (HSCs) and progenitors

(Weissman, 2000). Each blood cell, red blood cells, white blood cells, and platelets play

important roles in the body’s normal physiological functions. However, certain diseases and

conditions such as malaria, malnutrition, protozoan infections and pregnancy are among

various conditions that could disrupt normal haematopoiesis thus predisposing one to

anaemia. This is found to be more prevalent in both adults and children (FAO/WHO, 1988).

Anaemia results in the decrease of the oxygen- carrying capacity of the blood due to reduction

in circulating haemoglobin. The normal quantity of haemoglobin in humans is greater than 13

g/dL for males and 12 g/dL for females (Okochi et al., 2003). From epidemiological studies,

the World Health Organisation (WHO) estimates that in the year 2004, about two billion

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people, representing 30% of the world's population were anaemic (Adusi-Poku et al., 2008).

Also over 50% of pregnant women and over 40% of infants worldwide are anaemic with a

prevailing significant morbidity and mortality particularly in the developing world (Holden

and Acomb, 2007). Iron deficiency is the most common cause of nutritional anaemia which

affects over 600 million people throughout the world, particularly in developing countries

(Looker et al., 1997). The vulnerable groups are infants, young children and women of child-

bearing age (FAO/WHO, 1988). Hence anaemia is one of the leading health disorders posing

great threat to global healthcare. Medicinal plants are currently being used in various parts of

the world especially in the tropics for the treatment of various forms of anaemia. In South

Eastern Nigeria, with high prevalence of anaemia, aqueous decoctions of leaves of some

medicinal plants are very popular among village women for combating malaria induced

anaemia in children (Akah et al., 2009). Most vegetables and plants have been found to

contain haematinic agents such as folic acid, vitamin B6, iron which could stimulate the

erythropoietic pathway (Adedapo et al., 2002).

Moringa oleifera and Telfairia occidentalis leaves used as vegetables in various countries of

the world have been shown to have positive effects on some haematological parameters

(Alada, 2000; Adedapo et al., 2009). However, due to their ability to increase blood

parameters, their polyherbal formulations are been used in several localities among

housewives without any scientific investigation of their effect on haematological indices. It is

therefore expedient to compare the blood boosting capacities of the individual extracts with

their poyherbal formulation. This investigation will reveal their synergistic and antagonistic

potential as possible inclusions in infant weaning foods.

1.2 Moringa oleifera

Moringa (Moringa oleifera Lam) belongs to the Moringaceae family and has its origin in the

North-West region of India, South of the Himalayan Mountains (Makkar and Becker, 2007).

It is now widely cultivated and has been introduced in many locations in the tropics (Fahey et

al., 2001). There are about thirteen species of Moringa trees in the family Moringaceae but

Moringa oleifera is the most widely cultivated (Fuglie, 2000). This rapid-growing tree also

known as the horseradish, drumstick, benzolive, keolor, marango, malonge, moonga,

mulangay, nébéday, saijhan, sajna or Ben oil tree was utilized by the ancient Romans, Greeks

and Egyptians. It is a perennial softwood tree with timber of low quality, but which for

centuries has been advocated for traditional medicinal and industrial uses. It is an important

crop in India, Ethiopia, the Philippines and the Sudan and is grown in West, East and South

19

Africa, tropical Asia, Latin America, the Caribbean, Florida and the Pacific Islands. Moringa

oleifera commonly referred to as “miracle or wonder tree” (Fuglie, 2000) is also widely

cultivated in Nigeria for its medicinal values. The tree has the potential to improve nutrition,

boost food security and foster rural development. It also has significant socio-economic

importance because of its several nutritional, pharmacological (Caceres et al., 1991; Fuglie,

2000) and industrial applications (Makkar and Becker, 2007; Foidl et al., 2001). The leaves of

this plant contain a profile of important trace elements, and are also good sources of proteins,

vitamins, beta-carotine, amino acids and various phenolics (Anwar et al., 2007). 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, medication and industrial purposes (Khalafalla et al., 2010). People use

its leaves, flowers and fresh pods as vegetables, while others use it as livestock feed (Anjorin

et al., 2010).

1.2.1 Scientific classification of Moringa oleifera

Kingdom: Plantae

Division: Magnoliphyta

Class: Magnoliopsida

Order: Brassicales

Family: Moringaceae

Genus: Moringa

Species: oleifera

Kasolo et al. (2011)

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Plate1: The leaves of Moringa oleifera

Plate 2: The pods of Moringa oleifera

1.2.2 Medicinal, industrial and nutritional importance of Moringa oleifera

1.2.2.1 Medicinal uses of Moringa oleifera

Moringa preparations (e.g. extracts, decoctions, poultices, creams, oils, emollients, salves,

powders, porridges) have been used for the treatment and prevention of disease or infection

either through its dietary or topical administration (Palada, 1996). Seedpod extract of Moringa

has been shown to exact a dramatic reduction in skin Papilloma viruses in mouse model

21

(Bharali et al., 2003). It has also been shown that Moringa increases glucose tolerance in

diabetic patients (Ruckmani et al., 1998). The roots, seed, bark, leaves, fruit, flowers and

immature pods act as cardiac and circulatory drugs and possess antitumour activity

(Makonnen et al., 1997). Other medicinal uses of the plant include antifungal and

antispasmodic (Caceres et al., 1991). Moringa leaves contain flavonoids such as quercetin and

kaempferol which have been identified as the most potent antioxidants in the leaves

(Siddhuraju and Becker, 2003). Their antioxidant activities are higher than the conventional

antioxidants such as ascorbic acid, which is also present in large amounts in Moringa leaves

(Siddhuraju and Becker, 2003). This makes them effective against morphological changes and

oxidative damage by enhancing the activity of antioxidant enzymes, reducing the amounts of

lipid peroxidation and inhibiting free radical generation (Fahey et al., 2001). Some of the

compounds that have been isolated from Moringa preparations which are reported to have

hypotensive, anticancer and antibacterial activities include 4-(4'-O-acetyl-a-L-

rhamnopyranosyloxy) benzyl isothiocyanate, 4-(α-L-rhamnopyranosyloxy)benzyl

isothiocyanate, niazimicin, pterygospermin, benzyl isothiocyanate, and 4-(α-L-

rhamnopyranosyloxy) benzyl glucosinolate (Makonnen et al.,1997; Fahey et al., 2001).

1.2.2.2 Industrial uses of Moringa oleifera

Moringa oleifera has been employed in many industrial processes. The seed on extraction

yields 30-40% by weight of Moringa seed oil which is known as Ben oil. It is sweet, non-

sticking, non-drying oil that resists rancidity. The oil has been used in salad preparations, for

lubrication of machines and in the manufacture of perfume and hair care products (Tsaknis et

al., 1999). The seed cake obtained after extraction of the oil has been used as flocculent to

purify water, as fertilizer and as honey-and sugarcane juice clarifiers (Madsen et al., 1987).

Seeds of Moringa contain a glucosinolate that on hydrolysis yields 4-(α-L-rhamnosyloxy)-

benzyl isothiocyanate, an active bactericide and fungicide (Bosch, 2004). The leaves have

been used in the production of biogas, as domestic cleaning agent and in the production of

biopesticides. Tannins obtained from the bark of the tree are used for tanning hides and skins

(Fuglie, 2000). The seeds can also be used as a less expensive bio-absorbent for the removal

of heavy metals (Sharma and Clark, 1998). The cytokinine-type hormone extracts of Moringa

leaves in 80% ethanol as a foliar spray can be used to accelerate the growth of young plants

such as soyabean, blackbean, maize, onion, sorghum, tomato and sugar cane (Foidl et al.,

2001).

22

1.2.2.3 Nutritional uses of Moringa oleifera

Moringa oleifera have immense nutritional values as seen in its vitamin, mineral and amino

acid contents (Anwar et al., 2007). Nutritional evaluation of the plant according to Bosch

(2004), show that the leafy tips of Moringa oleifera contain per 100 g edible portion: water

(78.7 g), energy 268 kJ (64 kcal), protein (9.4 g), fat (1.4 g), carbohydrates (8.3 g), total

dietary fibre (2.0g), Ca (185 mg), Mg (147 mg), P (112 mg), Fe (4.0 mg), Zn (0.6 mg),

vitamin A (7564 IU), thiamine (0.3 mg), riboflavin (0.7 mg), niacin (2.2 mg), folate (40 µg),

and ascorbic acid (51.7 mg) while the raw fruits of the plant contain per 100 g edible portion:

water (88.2 g), energy 155 kJ (37 kcal), protein (2.1 g), fat (0.2 g), carbohydrates (8.5 g), total

dietary fibre (3.2 g), Ca (30 mg), Mg (45 mg), P (50 mg), Fe (0.4 mg), Zn (0.4 mg), vitamin A

(74 IU), thiamin (0.05 mg), riboflavin (0.07 mg), niacin (0.6 mg), folate (44 µg), and ascorbic

acid (141.0 mg), and as such could be a valuable source of nutrients for all age groups and

also could be used to combat malnutrition, especially among infants and nursing mothers

since they contain significant amounts of calcium, proteins and other vital elements for

growth and healthy development. Moringa oleifera has been shown to boost immune systems

(Olugbemi et al., 2010; Fuglie, 2000). The immature green pods which are also referred to as

drumsticks are probably the most valued and widely used part of the plant. They are

commonly prepared in a similar way as green beans and have a slight asparagus taste (Foidl et

al., 2001). The seeds are sometimes removed from mature pods and eaten as peas or roasted

like nuts. However, there could be considerable variations among the nutritional values of

Moringa, which depend on factors like genetic background, environment and cultivation

methods (Brisibe et al., 2009).

1.3 Telfairia occidentalis

Fluted pumpkin or Fluted guord (Telfairia occidentalis) is an edible vegetable plant that

belongs to the family Cucurbitaceae. It is a tropical vine grown mainly in West Africa for its

vegetable (Akoroda, 1990). In Nigeria, it is known locally as Ubong by Ibibios, Ugu by Igbos

and Iroko by Yorubas. The Ghananians refer to it as okrobonka while to the Siera Leoneans, it

is known as Oroko (Abiose, 1999).

The plant is dioeciously perennial and tolerant to drought. It has about 90 genera and more

than 700 species distributed all over the warm parts of the world (Purseglove, 1997). The crop

is grown across the low land humid tropics of West Africa and is partially drought-resistant

and tolerant to a wide range of soils (Okoli and Nyanayo, 1988). The fluted pumpkin is

planted solely from the seeds and although the plant grows all year round, it thrives best

23

during wet seasons and is usually harvested 120-150 days after planting (Okoli and Nyanayo,

1988). The young shoots, seeds and leaves of the female plant are the edible parts. The roots

of the plant are said to be poisonous (Abiose, 1999). Fluted pumpkin has remained a regional

treasure in West Africa (Abiose, 1999) where it is widely employed for its medicinal and

nutritional potentials. The crop is primarily grown as leafy vegetable and is used for human

consumption and animal fodder. The seed also has many nutritional and industrial uses

(Asiegbu, 1987; Akwaowo et al., 2000).

1.3.1 Scientific classification of Telfairia occidentalis

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Curcurbitalis

Family Cucurbitaceae

Genus Telfairia

Species occidentalis

Esuoso et al. (1998)

Plate 3: The leaves and pod of fluted pumpkin (Telfairia occidentalis)

24

1.3.2 The medicinal, industrial and nutritional importance of Telfairia occidentalis

1.3.2.1 Medicinal uses of Telfairia occidentalis

The pharmacological importance of this family of plants (Cucurbitaceae) is ample.

Considerable evidence from several epidemiological studies concerning the use of its

bioactive substances in a number of animal models, cell culture studies and clinical trials

validate its immense pharmacological activities (Gbile, 1986; Odoemena and Essien 1995;

Sofowora, 1996; Eseyin et al., 2000; Nwozo et al., 2004; Oboh et al., 2006). Telfairia

occidentalis is popularly used in ethnobotany as antidiabetic, antihypertensive, antitumouric,

antioxidant, immunodulator, antibacterial, antihypercholesterolemic, intestinal antiparasitic

and anti-inflammatory agent (Nwozo et al., 2004). Due to the presence of antioxidant and

antimicrobial properties, its minerals (especially Iron), vitamins (especially vitamin A and C)

and high protein contents (Kayode and Kayode, 2011), consumption of the leaves assist to

combat certain diseases. The aqueous extract of T. occidentalis has been shown to be

hepatoprotective against garlic-induced oxidative stress (Olorunfemi et al., 2005; Odoh, 2005)

while both aqueous and ethanolic extracts have demonstrated hypoglycaemic properties both

in normoglycaemic and alloxan-induced diabetic rats (Zhang, 2001; Zhang and Yao, 2002).

Studies have also shown the haematinic capacity of this plant hence the use of the concoction

of fresh leaves as a high-value health tonic for impotent men and a cheap and fast remedy for

acute anaemia (Ajibade et al., 2006; Kayode and Kayode, 2011).

1.3.2.2 Industrial uses of Telfairia occidentalis

Telfairia occidentalis provides an appreciable source of income for small farm families

(Akoroda, 1990). It has been utilized in many industrial processes. The fermented flour of

Telfairia cotyledons can be processed into seasonings, marmalade, high-protein infant

weaning food mixtures and different local products in West Africa (Egbekun et al., 1998).

The fluted pumpkin seed oil is composed of mainly unsaturated fatty acids which include:

oleic acid (39%), linoleic acid (28.3%) and other unsaturated fatty acids (Kayode et al., 1998).

The high unsaturated fatty acid content makes it a good source of cooking oil (Giami and

Isichei, 1999). The oil has also been shown to be a good source for the production of biodiesel

and soap production. The seed residue left after oil extraction is used as animal feeds.

Activated carbon produced from fluted pumpkin seed shell can be utilized for the removal of

lead II ion (Pb2+

) from simulated wastewater (Ejikeme et al., 2007). The roots have been

shown to have high concentrations of alkaloids, and the root extracts have been reported to be

25

poisonous to humans and animals and as such are used as rodenticides (Akoroda, 1990;

Schippers 2000).

1.3.2.3 Nutritional uses of Telfairia occidentalis

Fluted pumpkin contains naturally active components that comprise polysaccharides, fixed

oils, para-aminobenzoic acid, peptides, sterol, and proteins (Matsui et al., 1998; Appendino et

al., 1999; Kuhlmann et al., 1999). The fruits are a good source of carotenoid and γ -

aminobutyric acid (Arima and Rodriguez-Amaya, 1990; Gonzalez et al., 2001; Zhang, 2003).

The succulent tasty leaves, stems and seeds make fluted pumpkin one of the widely eaten

vegetables in homes and in restaurants across West Africa (Abiose, 1999). Fluted pumpkin

leaves are rich sources of protein, oil, vitamins, minerals, folic acid, calcium, zinc, potassium,

cobalt, copper, iron, vitamins A, C and K but low in crude fibre (Ladeji et al.,

1995; Ajibade et al., 2006). Relative to most vegetables, its protein content is very high

(Ladeji et al., 1995; Ajibade et al., 2006; Aregheore, 2007). Leaves of fluted pumpkin are

cheap source of nitrogen (Aregheore, 2007). According to Kayode and Kayode, 2011, leaves

of Telfairia occidentalis are rich in minerals, antioxidants, vitamins (such as thiamine,

riboflavin, nicotinamide and ascorbic acid. The young leaves also possess a high level of

magnesium (8.69 mg 100-1g) and iron (3.60 mg 100-1 g) (Akwaowo et al., 2000) and due to its

richness in iron and also an excellent proportion of essential amino acids to total nitrogen, the

leaves can be used to prevent and eliminate anaemia (Ajibade et al., 2006). Young shoots and

leaves are used for making soups for different kind of starchy dough and may be cooked alone

or in mixture with other vegetables. Immature seeds are usually preferred to mature ones and

are eaten cooked or roasted (Akwaowo et al., 2000). The nutritional value of the seeds (53%

fat and 27% crude protein) justifies their wide consumption (Agatemor, 2005). The seed

cotyledons due to their high protein content are processed into seasonings and are also utilised

for infant weaning foods (Agbede et al., 2008), bread flour supplement and in making

different local fermented foods “Ogiri ugu” (Egbekun et al., 1998; Giami and Isichei, 1999;

Giami et al., 2003; Christian, 2006). However, the usefulness of the seed as a source of

protein is limited by the presence of anti-nutrients such as phytic acid which have been

revealed to have harmful physiological properties on growing rats and chicks (Giami and

Isichei, 1999; Akwaowo et al., 2000). These anti-nutrients tend to lower the bioavailability of

minerals in humans and inhibit the digestibility of plant proteins (Lopez et al., 2002).

26

1.4 Phytochemicals

Phytochemicals are secondary metabolites produced by plants. They give plants their colour,

flavour, smell and are part of a plant’s natural defense system (Agte et al, 2000). These

compounds have been linked to human health by contributing to protection against

degenerative diseases (Anderson, 2004; Liu, 2004). Phytochemicals are present in a variety of

plants utilized as important components of both human and animal diets. These plants include

fruits, seeds, herbs and vegetables (Okwu, 2005). Epidemiological studies have shown that

the consumption of fruits and vegetables is associated with reduced risk of chronic diseases

(Doughari et al., 2009). Different mechanisms have been suggested for the action of

phytochemicals. They may act as antioxidants, or modulate gene expression and signal

transduction pathways (Doughari et al., 2009). They may be used as chemotherapeutic or

chemopreventive agents (D’Incalci et al., 2005). These bioactive compounds usually present

in relatively small quantities in higher plants, include the alkaloids, steroids, flavonoids,

terpenoids, tannins, saponins and many others.

1.4.1 Phytochemical constituents of plants

1.4.1.1 Terpenoids

Terpenoids, also known as isoprenoids are the major family of natural compounds,

comprising more than 40,000 different molecules (McCaskill and Croteau, 1998). The

isoprenoid biosynthetic pathway produces both primary and secondary metabolites that are of

great significance to plant growth and persistence (Haudenschild and Croteau, 1998)

Terpenoids are secondary metabolites that have molecular structures comprising carbon

backbones that are made up of isoprene (2-methylbuta- 1, 3-diene) units. The terpenoids

comprise of two isoprene units, containing ten carbon atoms. Among the primary metabolites

produced by this pathway are: the phytohormones- abscisic acid (ABA); gibberellic acid

(GAs) and cytokinins; the carotenoids; plastoquinones and chlorophylls involved in

photosynthesis; the ubiquinones required for respiration; and the sterols that impact

membrane structure (Harborne, 1998). Many of the terpenoids are important for the quality of

agricultural products, such as the flavour of fruits and the fragrance of flowers like linalool

(Pichersky et al., 1994). In addition, terpenoids may have medicinal properties such as anti-

carcinogenic (e.g. taxol and perilla alcohol), antimalarial (e.g. artemisinin), anti-ulcer,

antimicrobial or diuretic activity (e.g. glycyrrhizin) (Haudenschild and Croteau, 1998;

McCaskill and Croteau, 1998; Rodriguez-Concepcion, 2004; Bertea et al., 2005). The steroids

and sterols in animals are biologically produced from precursors of terpenoid and sometimes

27

terpenoids are added to proteins to increase their attachment to the cell membrane, a process

known as isoprenylation (Harborne, 1998).

1.4.1.2 Flavonoids

Flavonoids are polyphenolic compounds that are ubiquitous in nature and are categorized

according to their chemical structure into flavones, anthocyanidins, isoflavones, catechins,

flavonols, chalcones and flavanones (Dakora, 1995). They occur mostly in vegetables, fruits

and beverages like tea, coffee and fruit drinks. They accumulate in plants as phytoalexins

defending them against microbial attack (Grayer and Harborne, 1994; Harborne and Willams,

2000) and fungal attack (Jensen et al., 1998).

Flavonoids have been found to possess many useful effects on human health. They have been

shown to have several biological properties including anti-inflammatory activity, enzyme

inhibition, antimicrobial activity, oestrogenic activity (Havsteen, 1983; Harborne and

Willams, 2000), antioxidant and free-radical-scavenging ability (Robak and Gryglewski,

1988). Flavonoids have also been shown to interact with cytochrome P450 (Ng et al., 1996),

have anti-leukemic properties and mild vasodilatory properties useful for the treatment of

heart disease (Hodek, et al., 2002).

1.4.1.3 Saponins

Saponins are a group of secondary metabolites found widely distributed in the plant kingdom

as plant glycosides, characterized by a skeleton resulting from the 30-carbon precursor

oxidosqualene to which glycosyl residues are attached. They have sturdy foaming property

(Harborne, 1998) and are subdivided into triterpenoid and steroid glycosides. Saponins are

stored in plant cells as inactive precursors but are readily converted into biologically active

antibiotics by plant enzymes in response to pathogenic attack. (Okwu, 2005).

Saponins protect plants against attack by pathogens and pests (Price et al., 1978). These

molecules also have substantial marketable value and are processed as drugs and medicines,

foaming agents, sweeteners, taste converters and cosmetics (Hostettmann and Marston, 1995).

Saponin-containing plants are used as traditional medicines, especially in Asia, and are

intensively used in food, veterinary and medical industries (Hostettmann and Marston, 1995).

The pesticidal activity of saponins has long been reported (Irvine, 1961). Saponin-glycosides

are very lethal to cold-blooded organisms, but not to mammals (Hostettmann and Marston,

1995). Plant extracts containing a high percentage of saponins are commonly used in Africa to

treat water supplies and wells contaminated with disease vectors; after treatment, the water is

28

safe for human drinking (Hostettmann and Marston, 1995). Saponins induce a strong adjuvant

effect to T-dependent as well as T-independent antigens and also induce strong cytotoxic

CD8+ lymphocyte responses and potentiate the response to mucosal antigens (Kensil, 1996).

They have both stimulatory effects on the components of specific immunity and non-specific

immune reactions such as inflammation (de Oliveira et al., 2001) and monocyte proliferation

(Delmas et al., 2000).

Saponins have long been known to possess lytic action on erythrocyte cell membranes and

this property has been used in their detection. The haemolytic actions of saponins are alleged

to be due to their affinity for the aglycone moiety of membrane sterols, mainly cholesterol

with which they form undissolvable complexes (Glauert et al., 1962).

1.4.1.4 Tannins

Tannins are an exceptional group of water soluble phenolic metabolites of relatively high

molecular weight and having the ability to complex strongly with carbohydrates and proteins

(Petridis, 2010). Tannins are astringent, bitter plant polyphenols and the astringency from

tannins is what causes the dry and pucker feeling in the mouth following the consumption of

unripened fruit or red wine (Serafini et al., 1994). They are grouped into two forms-

hydrolysable and condensed tannins (Nityanand, 1997). Hydrolysable tannins are potentially

toxic and cause poisoning if large amounts of tannin-containing plant material such as leaves

of oak (Quercus spp.) and yellow wood (Terminalia oblongata) are consumed (Garg et al.,

1982) and as such seen as one of the anti-nutrients of plant origin because of their capability

to precipitate proteins, inhibit the digestive enzymes and decline the absorption of vitamins

and minerals (Khattab et al., 2010).

Several health benefits have been assigned to tannins and some epidemiological associations

with the decreased frequency of chronic diseases have been established (Serrano et al., 2009).

Several studies have shown significant biological effects of tannins such as antioxidant or free

radical scavenging activity as well as inhibition of lipid peroxidation and lipoxygenases in

vitro (Amarowicz et al., 2000). They have also been shown to possess antimicrobial, antiviral

antimutagenic and antidiabetic properties (Dolara et al., 2005). The antioxidant activity of

tannins results from their free radical and reactive oxygen species-scavenging properties, as

well as the chelation of transition metal ions that modify the oxidation process (Serrano et al.,

2009).

29

1.4.1.5 Steroids

Sterols are triterpenes which are based on the cyclopentanohydrophenanthrene ring system

(Harborne, 1998). Sterols in plants are generally described as phytosterols with three known

types occurring in higher plants: sitosterol (formerly known as β-sitosterol), stigmasterol and

campsterol (Harborne, 1998). These common sterols occur both as free and as simple

glucosides. Sterols have essential functions in all eukaryotes. Free sterols are integral

components of the membrane lipid bilayer where they play important role in the regulation of

membrane fluidity and permeability (Corey et al., 1993). While cholesterol is the major sterol

in animals, a mixture of various sterols is present in higher plants, with sitosterol usually

predominating. However, certain sterols are confined to lower plants such as ergosterol found

in yeast and many fungi while others like fucoterol, the main steroid of many brown algae is

also detected in coconut (Harborne, 1998).

1.4.1.6 Alkaloids

Alkaloids play a very important role in organism metabolism and functional activity. They are

metabolic products in plants, animals and micro-organisms. They occur in both vertebrates

and invertebrates as endogenous and exogenous compounds. Many of them have a disturbing

effect on the nervous systems of animals. Alkaloids are the oldest successfully used drugs

throughout the historical treatment of many diseases (Wink, 1998) and are one of the most

diverse groups of secondary metabolites found in living organisms. They have an array of

structural types, biosynthetic pathways, and pharmacological activities (Roberts and Wink,

1998). In plants and insects, toxic alkaloids are sequestered for use as a passive defense

mechanism by acting as deterrents for predating insects (Schmeller and Wink, 1998).

Alkaloids have been used throughout history in folk medicine in different regions around the

world. They have been a constituent part of plants used in phytotherapy. Many of the plants

that contain alkaloids are just medicinal plants and have been used as herbs. Some alkaloids

that have played an important role in this sense include aconitine, atropine, colchicine,

coniine, ephedrine, ergotamine, mescaline, morphine, strychnine, psilocin and psilocybin

(Schmeller and Wink, 1998).

Many alkaloids are known to have effect on the central nervous system and some act as anti-

paralytic (such as morphine, a pain killer). For example, quinine was widely used against

Plasmodium falciparum. In this respect, phytochemical screening reveals that most plants

traditionally used to treat malaria contain alkaloids among other things (Jeruto et al., 2011).

30

1.5 Haematopoiesis

Blood is a tissue which consists of fluid plasma in which are suspended a number of formed

elements (erythrocytes, leucocytes and thrombocytes). Its primary function is to provide a link

between the various organs and cells of the body, and to maintain a constant cellular

environment by circulating through every tissue, delivering nutrient to them and removing

waste products (Bowman and Rand, 1980). The functions of blood are made possible by the

individual and collective actions of its constituents; the biochemical and haematological

components. Generally, both the biochemical and haematological blood components are

influenced by the quantity and quality of feed and also the level of anti-nutritional elements or

factors present in the feed (Babatunde et al.,1992). The blood cells (erythrocytes, leucocytes

and thrombocytes) exist at fairly constant levels, suggesting the existence of feedback

mechanism for the cells (Guyton and Hall, 2006). All of the mature blood cells in the body are

generated from a relatively small number of hematopoietic stem cells (HSCs) and progenitors

(Weissman, 2000). HSCs or pleuriopotent stem cells are primarily found in the bone marrow.

Some are present in a variety of other tissues including peripheral blood and umbilical cord

blood. They are also found in low numbers in the liver, spleen, and many other organs

(Akashi et al., 1999). The process of blood formation and haemostasis is termed

haemopoiesis. In the foetus, haemopoiesis takes place in the yolk sac in the first trimester of

pregnancy and continues in liver and spleen in the fourth (4) month until the seventh (7)

month of intrauterine life. The bone marrow becomes the only haemopoietic organ from the

seventh (7) month of intrauterine life until birth (Akashi et al., 1999). After delivery and

throughout life, gradually red marrow is replaced by fatty yellow marrow at the ends of the

long and flat bones, e.g. skull, iliac bones, 25% of the cells in red marrow are erythrocyte

precursors while 75% are leucocyte precursors (Hoffbrand et al., 2006). The haematopoietic

system is an important index of physiological and pathological status in animals and man

since it is the first to become exposed to a high concentration of toxic agents (Babatunde et

al., 1992). Several other vitamins, mineral, amino acids and hormones are essential for normal

haematopoiesis.

1.5.1. Erythropoietin (EPO)

The stimulus for erythropoiesis is the hormone erythropoietin, secreted mostly by the kidney

although about 10% is produced in the liver (Hoffbrand et al., 2006). This hormone is not

preformed and stored in the body but stimulated by reduced oxygen (hypoxia) in the blood or

tissue. It’s mechanism of action is by triggering more of the pleuripotent stem cells

31

(haemocytoblast) to rapidly proliferate and follow the pathway for red blood cells production.

Increased red blood cells will lead to a rise in the blood oxygen levels, reducing

erythropoietin secretion by negative feedback mechanism (Hoffbrand et al., 2006).

1.5.2 Vitamin B12

Cobalamin, or vitamin B12 is the largest of the B complex vitamins, with a relative molecular

mass of over 1000. Vitamin B12 is found in foods of animal origin such as liver, fish and dairy

produce but does not occur in foods of plant origin. (Weir and Scott, 1998). In mammalian

cells, vitamin B12 is required by two enzymes; Methionine synthase and Methylmalonyl

coenzyme (CoA) mutase (Scott and Weir, 1994). Methionine synthase is required in folate

metabolism. It converts folate to tetrahydrofolate; a form usable by the body. Tetrahydrofolate

is required for DNA synthesis and vitamin B12 deficiency leads to impaired folate metabolism

and subsequently to impaired DNA synthesis. This affects rapidly dividing cells of the bone

marrow earlier than other cells, resulting in the production of large immature haemoglobin-

poor red blood cells (Shane, 2000).

The absorption of vitamin B12 is mediated by a glycoprotein, intrinsic factor secreted by the

parietal cells of the gastric mucosa, which also secretes hydrochloride acid (Bender, 2002).

Gastric acid and pepsin serve to release the vitamin from protein binding, and so make it

available (Bender, 2002).

1.5.3 Folic acid

Folic acid is the parent compound for a large group of compounds, the folates. Human do not

synthesize folates and as such require pre-formed folate as vitamin (Hoffbrand et al., 2006).

Folates are required in a variety of biochemical reactions in the body that require single

carbon unit metabolism as in amino acid interconversion (eg. Homocysteine conversion to

methionine) and also in the synthesis of purine precursors of DNA.

All body cells, including those of the bone marrow receive folate from the plasma as methyl

tetrahydrofolate (THF). Vitamin B12 is required for the demethylation of methyl THF, a

reaction in which homocysteine is methylated to methionine (Weir and Scott, 1998). THF is

the substrate for polyglutamates that act as coenzymes in a number of metabolic processes

e.g. 5, 10- methylene THF polyglutamate acts as a coenzyme for thymidylate synthetase, an

enzyme catalysing thymidylate synthesis, a rate-limiting step of DNA synthesis (Hoffbrand et

al., 2006). Although folate is widely distributed in foods, its deficiency is not uncommon. A

32

number of commonly used drugs cause folate depletion (Bender, 2002). Folate deficiency is

thought to cause megaloblastic anaemia by inhibiting thymidylate synthesis.

1.5.4 Iron

Iron is one of the most important elements involved in living process, which play central role

in almost all living cells both plants and animals. In animals, it is involved in oxygen transport

while in plant metabolism; iron is essential for photosynthetic and respiratory electron

transport, nitrate reduction, chlorophyll synthesis and detoxification of reactive oxygen

species (Achterberg et al., 2001). Iron functions as a cofactor for some enzymes and in its

ferrous state is required for the proper transport and storage of oxygen by means of

haemoglobin and myoglobin (Beard, 2001).

Iron is absorbed from the gastrointestinal tracts and transported in the blood bound to

transferrin. Transferrin delivers iron to tissues that have transferrin receptors particularly the

erythroblasts in the bone marrow which then incorporates the iron into haemoglobin (Beard,

2001). The oxygen-binding capacity of haemoglobin depends on the presence of the haem

group which has an atom of iron attached to four nitrogen atoms in the porphyrin ring.

Deficiency of iron leads to reduced synthesis of haemoglobin. Iron deficiency is the most

common cause of nutritional anaemia which affects over 600 million people throughout the

world, particularly in developing countries (Looker et al., 1997). Iron is also involved in

many other physiological functions in the body such as energy metabolism, gene regulation,

cell proliferation and differentiation, synthesis of both neurotransmitters and proteins (Beard,

2001). Other vitamins, amino acids and minerals essential for normal haemopoiesis include;

Vitamin C; which helps facilitate the absorption of iron from the gut and also in the normal

metabolism of vitamin B12 and folic acid, pyridoxine (vitamin B6); used for haem and

nucleoprotein production, vitamin E; for the synthesis of erythrocyte membrane. The amino

acids are needed for the synthesis of globulin protein. Trace elements such as copper and

cobalt stimulate the formation of erythropoietin (Cantor and Orkin, 2002).

1.6 Haematological indices

Blood parameters are the major indices of physiological, pathological, and nutritional status

of an organism and changes in the constituents of blood when compared to normal values

could be used to interpret the metabolic state of an animal (Babatunde et al., 1992; Maxwell

et al., 1990). It is one of the most sensitive targets for toxic compounds and an important

index of physiological and pathological status in man and lower animals (Mukinda and Syce,

33

2007). These parameters include red blood cells count, haemoglobin concentration, packed

cell volume, platelets count, total and differential white blood cells count and erythrocyte

sedimentation rate. A complete blood count (CBC), is a common blood test that provides

detailed information about the three types of cells in the blood: red blood cells, white blood

cells, and platelets. Each type of blood cell plays an important role in the body’s normal

function.

1.6.1 Red blood cell (RBC) count

Red blood cells are flexible biconcave disc that carry oxygen to tissues and remove waste

products from the body’s tissues. These cells contain haemoglobin, the molecule which

carries oxygen to the rest of the body. Red blood cells are 8µm in diameter. Their flexibility

allows them to pass repeatedly through the microcirculation whose minimum diameter is

3.5µm, to maintain haemoglobin in a reduced (ferrous) state (Hoffbrand et al., 2006). Red

blood cells are measured in millions per cubic milimetre (mil/uL) of blood.

1.6.2 Haemoglobin (HGB) value

Haemoglobin gives red blood cells their colour. Each red cell contains approximately 640

million haemoglobin molecules (Hoffbrand et al., 2006). Haemoglobin carries oxygen from

the lungs to the tissues and takes carbon dioxide (the waste products) from the tissues to the

lungs. Haemoglobin is measured in grams per decilitre (g/dL) of blood.

1.6.3 Packed Cell Volume or Hematocrit (HCT) value

The hematocrit or packed cell volume is the percentage of red blood cells in relation to the

total blood volume when blood is centrifuged at a constant speed and period of time.

1.6.4 Platelet count

Platelets help to stop bleeding and repair damage to the blood vessels by forming blood clots.

It results from a chemical “cascade” which begins with the prothrombin activators released

by platelets. Sometimes referred to as platelet thromboplastin, these chemicals cause the

macromolecule prothrombin to break down into smaller units including thrombin. Thrombin

acts on fibrinogen, a soluble polymer present in the plasma, and breaks it into monomers

which re-polymerize into insoluble fibrin. The fibrin forms threads which knit the platelets

and other cells into a clot. They are measured in thousands per cubic millimetre (m/uL) of

blood.

34

1.6.5 White blood cells (WBCs) count

White blood cells or leucocytes are the cells of the immune system. They defend the body

against pathogens, infection and foreign materials (Stock and Hoffman, 2000). White blood

cells are of two broad groups: the phagocytes and the immunocytes. The phagocytes include;

neutrophils, eosinophils, basophils as well as the monocytes. The immunocytes include the

lymphocytes, their precursor cells and plasma cells. Total WBC count is measured in

thousands per cubic mililitre (K/uL) of blood. However, the WBC count is not meaningful

unless the “differential” is also known. The differential count measures the percentage of

each of the five types of white blood cells:

• Neutrophils

Neutrophils are the most numerous white blood cells, making up about 65% of normal white

blood cell count. These cells, also called polymorphonuclear neutrophils (because of their

nuclear shape) are the most important phagocytic cell in the circulation (Hoffbrand et al.,

2006). The life span of these cells is between eight (8) to ten (10) days in the circulation and

usually makes their way to sites of infection where they engulf bacteria, viruses, infected cells

and debris.

• Basophils

They have many dark cytoplasmic granules which overlie the nucleus and contain heparin and

histamine. They are only seen occasionally in normal peripheral blood.

• Eosinophils

These cells are similar to neutrophils except that the nuclear lobes are rarely more than three.

The transit time of eosinophils in the blood is longer than for neutrophils. They enter

inflammatory exudates and a have special role in allergic responses, defence against parasites

and removal of fibrin formed during inflammation (Hoffbrand et al., 2006).

• Monocytes

These cells are usually large than other peripheral blood leucocytes and posses a large oval or

indented nucleus that obscures the cytoplasm. The cytoplasm contains many fine vacuoles

giving a ground-glass appearance.

• Lymphocytes

The lymphocytes are the immunological competent cells that assist the phagocytes in defence

of the body against infection and other foreign invasion. The immune response depends upon

two types of lymphocytes; B and T cells which are derived from the haematopieotic stem cell.

The B cells mature in the bone marrow and circulate in the peripheral blood while the T cells

35

develop from cells that have migrated to the thymus where they differentiates into mature T

cells (Hoffbrand et al., 2006).

Natural killer (NK) cells are another type of lymphocytes. They are large cells with

cytoplasmic granules and typically express surface molecules CD16 (Fc receptors), CD56 and

CD 57. They are designed to kill cells that have low level of expression of HLA class

molecules such as may occur during viral infection or on a malignant cell (Hoffbrand et al.,

2006).

Table 1 Normal ranges of the different blood cells

Hoffbrand et al. (2006)

1.7 The Liver

The liver is a self-regenerating organ that plays important roles in the body. It functions not

only in metabolism and removal of exogenous toxins and therapeutic agents responsible for

metabolic derangement but also in the biochemical regulation of fats, carbohydrates, amino

acids, protein, blood coagulation and immunomodulation (Ram and Goel, 1999). Due to its

ability to regenerate, even a moderate cell injury is not reflected by measurable change in its

metabolic function. However, damage caused by lipid peroxidation on the membrane of the

hepatocytes allows the leakage of some cytosolic enzymes of the liver into the blood stream.

1.7.1 Serum enzyme markers involved in hepatic disorder

When the integrity of the membrane of the hepatocytes is compromised, certain enzymes

located in the cytosol are released into the blood. Their estimation in the serum is a useful

36

quantitative marker for the evaluation of liver damage (Ram and Goel, 1999). Glutamate

dehydrogenase activity is not found in normal serum but moderate elevation is found in most

cases of acute hepatitis indicating cellular damage. Another demonstrable type of membrane

damage involves injury to lysosomes which leads to the release of acid ribonuclease and acid

phosphatises. Other cytosolic liver enzymes such alanine transaminase, aspartate transaminase

and alkaline phosphatise also leak into the blood stream when the membrane of the liver is

damaged. These enzymes are elevated to distinguish and assess the extent and type of

hepatocellular injury (Ram and Goel, 1999).

1.8 Aim of the Study

This study is aimed at investigating comparatively, the effects of aqueous extracts of the

leaves of Moringa oleifera and Telfairia occidentalis and the possible effect of the combined

aqueous extract on some biochemical and haematological parameters in Wistar rats.

1.9 Objectives of the research

• To determine qualitatively and quantitatively the phytochemical constituents of the

aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis.

• To determine the acute toxicity (LD50) of the aqueous extracts of leaves of Moringa

oleifera and Telfairia occidentalis.

• To determine the vitamin and mineral content of the aqueous extracts of leaves of

Moringa oleifera and Telfairia occidentalis.

• To determine the effects of Moringa oleifera and Telfairia occidentalis and their

combined aqueous leaf extracts on some liver marker enzymes.

• To determine the effect of Moringa oleifera and Telfairia occidentalis and their

combined aqueous leaf extracts on haematological parameters.

37

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 PLANT MATERIALS

Moringa oleifera leaves were obtained from Ovoko, Igbo-Eze LGA while the leaves of

Telfairia occidentalis were purchased from Ogige market in Nsukka LGA both in Enugu

State, Nigeria and were identified by Dr. U. Nzekwe of the Herbarium Unit of the Department

of Plant Science and Biotechnology, University of Nigeria, Nsukka

2.1.2 ANIMALS

Fifty-four (54) weanling Wistar rats of approximately three (3) weeks old and weighing

between 50-80g were used for the study. The rats were obtained from the Animal House of

the Department of Zoology and Environmental Biology, University of Nigeria, Nsukka. The

rats were fed with rat pellets and water ad libitum

2.1.3 EQUIPMENT

The equipment used were obtained from the Departments of Biochemistry and Veterinary

Medicine, University of Nigeria, Nsukka. They include:

Centrifuge PAC, Pacific

Glasswares Pyrex

Haematocrit centrifuge Hawksley, Germany

Micropipette Perfect

Microscope Calstock, Germany

Naubeur chamber and counter R & S Scientific Inc, India

Refrigerator Thermocool

Spectrophotometer Spectronic 20D, USA Syringe Lifescan

Thermometer Zeal

Water bath Gallenkamp, England

Weighing balance Mettler HAS, USA

2.1.4 CHEMICALS AND REAGENTS

The chemicals and reagents used were of analytical grade. They include:

Absolute ethanol BDH, England

Acetone Sigma, USA

38

Aluminium chloride BDH, England

Bismuth carbonate BDH, England

Ethyl acetate BDH, England

Fehling’s solutions A and B BDH, England

Ferric chloride Merck, USA

Ferrous sulphate BDH, England

Glacial acetic acid Sigma, USA

Hydrochloric acid BDH, England

Lead acetate BDH, England

Millon’s reagent BDH, England

Mercuric chloride Sigma, USA

Naphthylene diamine dihydrochloride BDH, England

Olive oil Goya, Spain

Picric acid Lab Tech Chemicals

Potassium dihydrogen sulphate BDH, England

Potassium iodide East Anglia, England

Potassium sulphate May & Baker, England

Potassium hydroxide May & Baker, England

Potassium chloride Merck, USA

Phosphoric acid Sigma, USA

Saponin Lab Tech Chemicals

Sulphuric acid BDH, England

Tannic acid Sigma, USA

α –naphthol Sigma, USA

2.2 METHODOLOGY

2.2.1 PREPARATION OF PLANT EXTRACTS

Known weights (200g) of fresh leaves of Moringa oleifera and Telfairia occidentalis

separated from the stem, were washed with clean water to remove dirt and sand, drained, and

chopped. They were macerated in 500ml of tap water and then filtered to obtain homogenous

aqueous extracts while the combined extract was prepared by mixing equal volumes of the

different extracts in the ratio of 1:1 (v/v). The extracts were prepared at three-day interval for

the period of the experiment .

39

2.2.2 PREPARATION OF REAGENTS FOR PHYTOCHEMICAL ANALYSIS

5% (w/v) Ferric chloride solution

A quantity (5.0g) of ferric chloride was dissolved in 100ml of distilled water.

Ammonium solution

A quantity (187.5ml) of the stock concentrated ammonium solution was diluted in 31.25ml of

distilled water and then made up to 500ml with distilled water.

45% (v/v) ethanol

Absolute ethanol (45ml) was mixed with 55ml of distilled water.

Aluminium chloride solution

Aluminium chloride (0.5g) was dissolved in 100ml of distilled water.

Dilute sulphuric acid

Concentrated sulphuric acid (10.9ml) was mixed with 5ml of distilled water and made up to

100ml.

Lead acetate solution

A quantity (45ml) of 15% lead acetate (i.e. 15.0g of lead acetate in 100ml of distilled water)

was dissolved in 20ml of absolute ethanol and made up to 100ml with distilled water.

Wagner’s reagent

Iodine crystals (2.0g) and 3.0g of potassium iodide were dissolved in minimum amount of

water and then made up to 100ml with distilled water.

Mayer’s reagent

A known weight (13.5g) of mercuric chloride was dissolved in 50ml of distilled water. Also,

5.0g of potassium iodide was dissolved in 20ml of distilled water. The two solutions were

mixed and the volume made up to 100ml with distilled water.

Dragendorff’s reagent

A quantity (0.85g) of bismuth carbonate was dissolved in 100ml of glacial acetic acid and

40ml of distilled water to give solution A. Another solution called solution B was prepared by

dissolving 8.0g of potassium iodide in 20ml of distilled water. Both solutions were mixed to

give a stock solution.

Molisch reagent

A quantity (1.0g) of α-naphthol was dissolved in 100ml of absolute ethanol.

2% (v/v) Hydrochloric acid

40

Concentrated hydrochloric acid (2.0ml) was diluted with some distilled water and made up to

100ml.

1% (w/v) Picric acid

A quantity (1.0g) of picric acid was dissolved in 100ml of distilled water.

2.3 Qualitative phytochemical analysis of Moringa oleifera and Telfairia occidentalis

The phytochemical analysis of the leaves of Moringa oleifera and Telfairia occidentalis was

carried out according to the method of Harborne (1998) and Trease and Evans (1983). The

methods are shown below:

2.3.1 Test for alkaloids

The sample (0.2g) was boiled with 5ml of 2% HCl on a steam bath. The mixture was filtered

and 1ml of the filtrate was treated with 2 drops of the following reagents

(i) Dragendorff’s reagent: An orange precipitate indicates the presence of alkaloids.

(ii) Mayer’s reagent: A creamy-white precipitate indicates the presence of alkaloids.

(iii) Wagner’s reagent: A reddish-brown precipitate indicates the presence of alkaloids.

(iv) Picric acid (1%): A yellow precipitate indicates the presence of alkaloids.

2.3.2 Test for flavonoids

A quantity (0.2g) of the sample was heated with 10ml ethyl acetate in boiling water for 3

minutes. The mixture was filtered, and the filtrate was used for the following tests.

(i) Ammonium test: 4ml of the filtrate was shaken with 1ml of dilute ammonium solution

to obtain two layers. The layers were allowed to separate. A yellow precipitate observed in the

ammonium layer indicates the presence of flavonoids.

(ii) Aluminium chloride test: 4ml of the filtrate was shaken with 1ml of 1% aluminium

chloride solution and observed for light yellow colouration that indicates the presence of

flavonoids.

2.3.3 Test for glycosides

A known weight of the sample (0.2g) was mixed with 30ml of distilled water and 15ml of

dilute sulphuric acid respectively and heated in a water bath for 5minutes. The mixtures was

filtered and the filtrates used for the following test.

(i) To 5ml of each of the filtrate, 0.3ml of Fehling’s solutions A and B was added until it

turned alkaline (tested with litmus paper) and heated on a water bath for 2 minutes. A brick-

red precipitate indicates the presence of glycosides.

41

2.3.4 Test for proteins

Distilled water (5ml) was added to 0.1g of the sample. The mixture was left to stand for 3

hours and then filtered. To 2ml portion of the filtrate was added 0.1ml of Millon’s reagent.

The mixture was shaken and kept for observation. A yellow precipitate indicates the presence

of proteins.

2.3.5 Test for carbohydrates

A known weight (0.1g) of the sample was shaken vigorously with water and filtered. To the

aqueous filtrate was added few drops of Molisch reagent followed by vigorous shaking again.

Then, 1ml of concentrated sulphuric acid was carefully added down the side of the test tube to

form a layer below the aqueous solution. A brown ring at the interface indicates the presence

of carbohydrates.

2.3.6 Test for reducing sugars

A known weight (0.1g) of the sample was shaken vigorously with 5ml of distilled water and

filtered. To the filtrate was added equal volumes of Fehling’s solutions A and B and shaken

vigorously. A brick-red precipitate indicates the presence of reducing sugars.

2.3.7 Test for saponins

A quantity (0.1g) of the sample was boiled with 5ml of distilled water for 5 minutes. The

mixture was filtered while still hot. The filtrate was used for the following tests.

(i) Emulsion test: A quantity, 1ml of the filtrate was added to two drops of olive oil. The

mixture was shaken and observed for the formation of emulsion.

(ii) Frothing test: A quantity, 1ml of the filtrate was diluted with 4ml of distilled water.

The mixture was shaken vigorously and then observed on standing for a stable froth.

2.3.8 Test for tannins

A quantity (2g) of the sample was boiled with 5ml of 45% ethanol for 5 minutes. The mixture

was cooled and then filtered and the filtrate was treated with the following solutions.

(i) Lead acetate solution: To 1ml of the filtrate, 3 drops of lead acetate solution was

added. A gelatinous precipitate indicates the presence of tannins.

(ii) Bromine water: To 1ml of the filtrate was added 0.5ml of bromine water and then

observed for a pale brown precipitate.

42

(iii) Ferric chloride solution: a quantity (1ml) of the filtrate was diluted with distilled water

and then 2 drops of ferric chloride solution was added. A transient greenish to black colour

indicates the presence of tannins.

2.3.9 Test for oils

A quantity (0.1g) of the sample was pressed between filter papers and the papers observed.

Translucency of the filter paper indicates the presence of oils.

2.3.10 Test for resins

(i) Precipitate test: A quantity, 0.2g of the sample was extracted with 15ml of 96% ethanol.

The alcoholic extract was poured into 20ml of distilled water in a beaker. A precipitate

occurring indicates the presence of resins.

(ii) Colour test: A quantity, 0.12g of the sample was extracted with chloroform and

concentrated to dryness. The residue was re-dissolved in 3ml of acetone, and 3ml of

concentrated HCl added and heated in a water bath for 30minutes. A pink colour which

changes to magenta indicates the presence of resins.

2.3.11 Test for terpenoids and steroids

About 9ml of ethanol was added to 1g of the sample and refluxed for a few minutes and

filtered. The filtrate was concentrated to 2.5ml on a boiling water bath, and 5ml of hot water

was added. The mixture was allowed to stand for 1hour, and the waxy matter filtered off. The

filtrate was extracted with 2.5ml of chloroform using a separating funnel. To 0.5ml of the

chloroform extract in a test tube was carefully added 1ml of concentrated sulphuric acid to

form a lower layer. A reddish-brown interface shows the presence of steroids.

Another 0.5ml aliquot of the chloroform extract was evaporated to dryness on a water bath

and heated with 3ml of concentrated sulphuric acid for 10 minutes on water. A grey colour

indicates the presence of terpenoids.

2.4 Quantitative phytochemical analysis of Moringa oleifera and Telfairia occidentalis

2.4.1 Alkaloid determination

The determination of alkaloids was done as described by Harborne (1998). A portion (5 g) of

the sample was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was

added, covered and allowed to stand for 2 h. This was filtered and the extract was

concentrated on a water bath to one – quarter of the original volume. Concentrated

43

ammonium hydroxide was added drop-wise to the extract till a precipitate was formed. The

precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The

residue was the alkaloid, which was dried and weighed.

2.4.2 Determination of Flavonoids

This was done according to the method of Harborne (1998). A quantity, 5 g of the sample was

boiled in 50ml of 2MHCl solution for 30min under reflux. It was allowed to cool and then

filtered through whatman No. 1 filter paper. A measured volume of the extract was treated

with equal volume of ethyl acetate starting with drop. The solution was filtered into a weighed

crucible. The filtrate was heated to dryness in an oven at 60 C. the dried crucible was weighed

again and the difference in the weight gave the quantity of flavonoid present in the sample.

2.4.3 Determination of Steroids

This was done by the method described by Okeke and Elekwa (2003). A known weight of

each sample was dispersed in 100ml freshly distilled water and homogenized in a laboratory

blender. The homogenate was filtered and the filtrate was eluted with normal ammonium

hydroxide solution (pH 9). The eluate (2ml) was put in test tube and mixed with 2ml of

chloroform. Ice-cold acetic anhydride (3ml) was added to the mixture in the flask and 2 drops

of conc. H2SO4 were cautiously added. Standard sterol solution was prepared and treated as

described above. The absorbances of standard and prepared sample were measured in a

spectrophotometer at 420 nm.

2.5 Determination of antinutrient contents of Moringa oleifera and Telfairia occidentalis

2.5.1 Tannins

The method of Swain (1979) was used for the determination of the tannin contents of

Moringa oleifera and Telfairia occidentalis. A quantity (0.2 g) of finely ground sample was

measured into a 50 ml beaker. About 20 ml of 50% methanol was added and covered with

paraffin and placed in a water bath at 77-80 0C for 1 hr and stirred with a glass rod to prevent

bumping. The extract was filtered using a double layer of Whatman No. 1 filter paper into a

50 ml volumetric flask then 20 ml distilled water, 2.5 ml Folin-Denis reagent and 10 ml of

17% Na2CO3 were added and mixed properly. The mixture was made up to mark with

distilled water and allowed to stand for 20 mins when a bluish-green colouration developed.

Standard tannic acid solutions of range 0-10 ppm were treated similarly as 1ml of sample

44

above. The absorbances of the tannic acid standard solutions as well as samples were read

after colour development at 760 nm. The tannin content was calculated using the formula:

Tannin (%) = Absorbance of sample x Average gradient x Dilution factor Weight of sample x 10000

2.5.2 Cyanogenic glycoside

The extraction was according to the method of Wang and Filled as described by Onwuka

(2005). A portion (5 g) of sample was made into paste and dissolved in 50 ml distilled water.

The extract was filtered and the filtrate was used for cyanide determination. To 1 ml of the

sample filtrate, 4 ml of alkaline picrate was added and absorbance was read at 550 nm and

cyanide content was extrapolated from a cyanide standard curve.

Cyanide (mg/g) = Absorbance x GF x DF Sample weight Where: GF = gradient factor and DF = dilution factor.

2.6 Acute toxicity test of aqueous extracts of the leaves of Moringa oleifera and

Telfairia occidentalis

The method of Lorke (1983) was used for the acute toxicity test of the aqueous extracts of

leaves of Moringa oleifera and Telfairia occidentalis. Thirteen (13) albino mice were utilized

in this study. The test involved two stages. In stage one, the animals were grouped into three

(3) groups of three rats each and were given 10, 100 and 1000mg/kg body weight of the

extracts respectively. The second stage involved the number of death that occurred in the

different groups in stage one.

2.7 Determination of the vitamin and mineral contents of the aqueous leaf extracts of

Moringa oleifera and Telfairia occidentalis

The vitamin and mineral contents were determined using the methods of Pearson (1976) and

AOAC (2006). The details are outlined below:

2.7.1 Vitamin B6

The sample (1g) was macerated with 50ml of distilled water. The mixture was filtered. To

1ml of the filtrate, 2ml of distilled water was added. Sodium acetate (0.4ml), diazotised

reagent and 0.2ml of sodium carbonate. The mixture was vortexed and absorbance taken at

540nm. The concentration of vitamin B6 was extrapolated from the standard curve of vitamin

B6.

45

2.7.2 Folic acid

A quantity (2g) of the sample was macerated with 20ml of 3% K2 PO4. The mixture was

filtered and the absorbance of the filterate taken at 256nm. The concentration of the folic acid

in the sample was extrapolated from the standard curve of folic acid.

2.7.3 Iron

About 5.0 g of sample was placed in a clean dried crucible. The sample was ashed in a muffle

furnace for 3hours at 600oC. It was cooled and to it 5 ml of 30% HCl and 10ml of distilled

water were added. The mixture was transferred to a 50 ml volumetric flask and made up to

volume with distilled water. To 5 ml of the sample in a 25 ml volumetric flask, quinol

solution (1 ml), 2% phenonthroline solution (3 ml) and sodium citrate solution (5 ml) were

added. The mixture was made up to 25 ml with diluted water and allowed to stand for 4

hours. Absorbance was taken at 510 nm. The standard solutions were prepared similarly and

the concentration of iron extrapolated from the standard curve.

2.8 Experimental Design

Fifty-four (54) weanling Wistar rats were used for the study. They were acclimatized for

seven days with free access to feed and water. After acclimatization, they were randomly

distributed into six (6) groups of nine rats each. The baseline haematological parameters were

determined before the administration of the extracts. The treatment lasted for 14 days in

which analyses were done on day 7 and day 14. The route of administration was oral with the

aid of an intubation tube. The groups and doses administered are summarised below.

Group 1 (control group) was given tap water

Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves

Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves

Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves

Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves

Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera

leaves (1:1 v/v)

2.9 Haematological parameters of rats treated with aqueous leaf extracts of Moringa

oleifera and Telfairia occidentalis

All the haematological indices were assayed by the method outlined by Dacie and Lewis

(2001).

46

2.9.1 Determination of Packed Cell Volume

Principle: When whole blood sample is subjected to a centrifugal force for maximum RBC

packing, the space occupied by the RBCs is measured and expressed as percentage of the

whole blood volume.

Method: Using microhaematocrit method, a well-mixed anticoagulated whole blood was

allowed to enter capillary haematocrit tubes until they were approximately 2/3 filled with

blood. Blood filling was done for each tube. One end of each tube was sealed with plastacine

and placed in the medial grooves of the haematocrit centrifuge head exactly opposite each

other, with the sealed end away from the centre of the centrifuge. All tubes were spun for five

minutes at 1000rpm. The tubes were removed as soon as the centrifuge had stopped spinning.

Calculation: PCV was obtained for each tube using microhaematocrit-reader by measuring

the height of the RBC column and expressing this as a ratio of the height of the total blood

column.

PCV (%) = Height of cell column x 100 Height of total blood column

2.9.2 Determination of Haemoglobin (Hb) Concentration

Principle: When whole blood is added to Drabkin’s reagent: a solution containing KCN and

K3Fe(CN)6, KCN converts Hb-Fe2+ (ferrous) to Hb-fe3+ (ferric) state to form methaemoglobin

which then reacts with KCN to form a stable pigment, cyanmethaemoglobin complex. The

colour intensity of this mixture is measured in a spectrophotometer at a wavelength of 540nm

(or using a yellow-green filter). The optical density (OD) of the solution is proportional to the

haemoglobin concentration. All forms of Hb (Hb-C, Hb-O, etc) except Hb-S are measured

with this cyanmet-method.

Method: Exactly 5.0ml of Drabkin’s reagent was pipetted into two test tubes 1 and 2 and a

well-mixed sample of EDTA blood (0.02ml) was pipetted into the tubes, rinsing the pipette

five times with the reagent, until all the blood was removed from the pipette. The solutions

were well mixed and allowed to stand at 250˚C for 10 mins in order to allow the formation of

Cyan-met-haemoglobin. The mixtures were transferred into cuvettes and read in a

spectrophotometer at a wavelength of 540nm. The Drabkin’s reagent in tube 1 was used as the

blank (setting the percentage transmittance at 100 %). The readings from each tube was

recorded and the actual Hb values in g/dl were determined from a pre-calibrated chart.

47

Calculation:

Hb in g/dl = Absorbance of test × Conc. of standard (in mg/dl) Absorbance of standard

2.9.3 Determination of Red Blood Cells (RBCs)

Principle: When whole blood is diluted with an isotonic fluid, it prevents lysis and facilitates

counting of the red cells. Some isotonic solutions in use include Hayem’s solution, Gower’s

solution or 0.85% NaCl solutions.

Method: Using the Thoma (manual counting) method, anticoagulated blood was drawn up to

the 0.5ml mark in the RBC count pipette and diluted to a 101 mark with RBC diluting fluid

(1: 200 dilution). Dilution was repeated with the replicate tube. Counting chamber was

cleaned; both pipettes were shaken three times; counting chamber filled (first expelling the

first 4 drops of the mixture), allowing approximately three minutes for the RBCS to settle.

Red cells were counted using the counting steps as follows:

1. The filled counting chamber was carefully placed on the microscope stage.

2. Using low power (x10 objective) the large centre square was placed in the middle of

the field of vision and the entire large square was carefully examined for even distribution of

RBCs.

3. The high-dry objective was carefully changed, moving the counting chamber so that

the small upper left corner square (this square is further sub-divided into 16 even smaller

squares) is completely in the field of vision.

4. All the RBCs were counted in the squares, also counting the cells on the two of the

margins but excluding those lying on the other two sides.

Calculation:

The RBCs (in mm3) = cells counted × correction for volume × correction for dilution

= RBCs counted in 5 small squares × 200 × 1.0/0.2 (or 50)

= number of RBCS counted in five squares ×104

2.9.4 Determination of White Blood Cells (WBCs)

Principle: When whole blood is mixed with weak acid solution, it dilutes the blood and

haemolyses the RBCs, enabling the WBCs to be counted.

Method: Manual WBC counting method was used as follows.

48

Dilution of Blood:

(a) The blood specimen was mixed approximately for one minute; using the aspirator and

WBC pipette, blood was drawn to the 0.5 mark in the pipette.

(b) Blood was removed from the outside of the pipette with clean gauze.

(c) Holding the pipette almost vertically, the tip was placed into the counting diluting

fluid to draw it slowly. While gently rotating the pipette, to ensure proper mixing, the diluting

fluid was aspirated until it reached the 11 mark.

(d) The pipette was placed in a horizontal position and firmly holding the index finger of

either hand over the opening in the tip of the pipette, aspirator was detached from the other

end of the pipette. This is 1:20 dilution.

(e) Having now completed the dilution of blood, the counting chamber and cover glass

were cleaned with a lint-free cloth.

Filling the counting chamber: Approximately 0.02ml of well mixed EDTA- anticoagulated

venous blood sample was added to 0.38ml of diluted fluid and dispensed into a small

container. One of the grids of the counting chamber was filled with re-mix of the diluted

blood sample using a Pasteur pipette, taking care not to overfill the area. The filled area was

left undisturbed for two minutes to allow time for the white blood cells to settle, after which

the underside of the chamber was dried and placed on the microscope stage.

Counting the white blood cells: Using the x10 objective, with the condenser iris closed

sufficiently to give good contrast, the ruling of the chamber and white cells were focused until

the cells appeared as small black dots. The cells in the four large squares of the chamber were

then squarely counted.

Calculation:

• The number of white cells per litre of blood was calculated as follows:

• The total number of cells counted was divided by 2

• The number obtained was then divided by 10

• The result was then multiplied by 109 to give the white cell count.

2.9.5 Determination of differential white blood cell counts

Principle: Wright’s stain is a polychromatic stain in that the dye present in the stain produces

multiple colours when applied to cell, to enable differential identification and counting.

49

Method: Thin blood films were made on slides and allowed to air- dry. The blood smears

were fixed by flooding with methanol after which the slides were flooded with Wright’s stain

and after four minutes mixed with an equal volume of phosphate buffer and allowed to stand

for seven minutes. The slides were rinsed using distilled water and stood up on one end to dry.

Counting the differential white blood cells: The stained blood smears were examined using

the x10 objective lens. A drop of oil immersion was placed on the slides and counting began

in areas where RBCs were slightly overlapping; moving the slides, each white cell was

counted and recorded on a differential cell counter until 100 white blood cells have been

counted (Monocyte, lymphocytes, neutrophils, basophils and eosinophils).

2.10 Liver function test of rats fed with aqueous extract of Moringa oleifera and

Telfairia occidentalis

2.10.1 Determination of Alanine Aminotransferase (ALT)

Principle: ALT is measured by monitoring the concentration of pyruvate hydrazone formed

with 2, 4-dinitrophenylhydrazine. The colour intensity is measured against the blank at

540nm.

Method: The blank and sample test tubes were set up in duplicates. Serum (0.1ml) was

pipetted into the sample tubes. To these were added 0.5ml buffer solution containing

phosphate buffer, L-alanine and α-oxoglutarate. The mixtures were thoroughly mixed and

incubated for exactly 30 minutes at 37 0C and pH 7.4. Reagent containing 2, 4-

dinitrophenylhydrazine (0.5ml) was later added to both tubes while 0.1ml of sample was

added to sample blank tube. The tubes were mixed thoroughly and incubated for exactly 20

minutes at 25 0 C. Sodium hydroxide (5.0ml) solution was then added to each tube and mixed.

The absorbance was read against the blank after 5 minutes at 540nm.

Calculation: The activity of ALT was read up from Table 2 (see Appendix).

2.10.2 Determination of Aspartate Aminotransferase (AST)

Principle: AST or SGOT is measured by monitoring the concentration of oxaloacetate

hydrazone formed with 2, 4-dinitrophenylhydrazine. The colour intensity is measured against

the blank at 546nm.

Method: The blank and sample test tubes were set up in duplicates. Serum (0.1ml) was

pipetted into the sample tubes. Reagent 1 (0.5ml) was pipetted into both sample and blank

tubes. The mixtures were thoroughly mixed and incubated for exactly 30 minutes at 37 0C and

pH 7.4. Reagent 2 (0.5ml) containing 2, 4-dinitrophenylhydrazine was added into all the test

50

tubes followed by 0.1ml of sample into the blank tubes. The tube contents were mixed

thoroughly and incubated for exactly 20 minutes at 25 0 C. Sodium hydroxide (5.0ml) solution

was then added to each tube and mixed. The absorbance was read against the blank after 5

minutes at 546nm.

Calculation: The activity of AST was read up from Table 3 (see Appendix).

2.10.3 Determination of Alkaline phosphatase (ALP)

Principle: The principle of this method is based on the reaction involving serum alkaline

phosphatase and a colourless substrate of phenolphthalein monophosphate. The ALP

hydrolyses phenolphthalein monophosphate giving rise to phosphoric acid and

phenolphthalein which at alkaline pH values, turn pink that can be determined

spectrophotometrically.

P-nitrophenylphosphate + H20 --------� ALP ------� PO42-

+ P-nitrophenol (pink at pH=9.8)

Method: The blank and sample test tubes were set up in duplicates. Sample (0.05ml) was

pipetted into the sample test tubes while 0.05ml of distilled water was pipetted into the blank

tube. Substrate (3.0ml) was pipetted into each tube respectively, which was then mixed and

the initial absorbance taken at 405nm. The stop watch was started and the absorbance of the

sample and the blank read again three more times at one minute intervals.

Calculation: alkaline phosphatase activity was calculated as follows:

Activity of ALP (in U/L) = Absorbance of sample x 3300

Absorbance of standard

2.11 Statistical Analysis

The data obtained were expressed as mean of 3 replicates ±SD. Statistical analysis was carried

out using the Statistical Package for Social Sciences (SPSS) version 19. Two-way and one-

way analyses of variance were adopted for comparison, and the results were subject to post

hoc test using least square deviation (LSD). p <0.05 were considered significant for all the

results.

51

CHAPTER THREE

RESULTS

3.1 Phytochemical analysis of the leaves of Moringa oleifera and Telfairia occidentalis.

The phytochemical constituents of both Moringa oleifera and Telfairia occidentalis are shown

in Table 4.

Table 4: Qualitative phytochemical analysis of the aqueous leaf extracts of Moringa

oleifera and Telfairia occidentalis.

Phytochemicals Moring

oleifera

Telfairia

occidentalis

Alkaloids +++ +++

Flavonoids +++ +++

Steroids + +

Saponins + +++

Glycosides ++ -

Terpenoids ++ +

Resins - -

Reducing sugars - -

Carbohydrates +++ ++

Proteins +++ ++

Tannins +++ ++

Fats and oils +++ +

+ Slightly present ++ Moderately present +++ Highly present

– Not present

52

3.2 Quantitative phytochemical constituents of aqueous leaf extracts of Moringa oleifera

and Telfairia occidentalis.

Table 5 shows the phytochemical contents of the aqueous leaf extracts Moringa oleifera and Telfairia occidentalis.

Table 5: Quantitative phytochemical contents of the aqueous leaf extracts of Moringa

oleifera and Telfairia occidentalis

Phytochemical constituents Moringa oleifera Telfairia occidentalis

Alkaloids (mg/g) 21.53 ± 0.53 12.15 ± 0.42

Flavonoids (mg/g) 15.42 ± 0.10 18.50 ± 0.14

Steroids (mg/g) 15.28 ± 0.16 19.92 ±0.55

Tannins (mg/g)

Saponin (%)

Cyanogenic glycoside (mg/g)

7.79 ± 0.05

0.59± 0.01

0.03± 0.00

5.58 ± 0.02

0.52± 0.00

0.00± 0.00

53

3.3 Acute toxicity (LD50) of the aqueous leaf extracts of Moringa oleifera and Telfairia

occidentalis.

The acute toxicity test of the aqueous extracts of Moringa oleifera and Telfairia occidentalis

as shown in Tables 6 and 7 (see appendix II) shows that no deaths were recorded in the mice

up to 5000mg/kg body weight of the extracts though the animals showed signs of toxicity at

this concentration of the extracts within 24 hours of constant observation.

54

3.4 Vitamin and mineral contents of Moringa oleifera and Telfairia occidentalis leaves.

The leaves of both Moringa oleifera and Telfairia occidentalis are shown in Table 8 to

contain some vital vitamin and minerals. The table further reveals that the iron contents of

both Moringa oleifera and Telfairia occidentalis leaves were significantly different (p< 0.05).

Hence the iron content significantly decreased (p< 0.05) in Moringa oleifera when compared

to Telfairia occidentalis.

Table 8: Vitamin and mineral constituents of Moringa oleifera and Telfairia occidentalis

Vitamin/Mineral

constituents

Moringa oleifera

Mean±SD

Telfairia occidentalis

Mean±SD

Iron(mg/g) 0.70±0.58 1.38±0.18

Folic(mg/g) 0.37±0.06 0.26±0.01

Vitamin B6(mg/g) 0.84±0.00 0.88±0.35

55

3.5 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

haemoglobin concentration.

Fig. 1 shows the haemoglobin concentration of rats fed aqueous extracts of Moringa oleifera

and Telfairia occidentalis leaves. No significant (p> 0.05) increase was observed in the

haemoglobin concentrations of the treatment groups (Groups 2, 3, 5 and 6) except group 4

(treated with 20ml/kg body weight of aqueous extract of T. occidentalis extract) when

compared with the control (group 1), during the first 7 days of treatment. After 14 days of

treatment with both extracts of M. oleifera and T. occidentalis, a significant (p< 0.05) increase

was observed in groups 3 and 4 when compared the group1 (control). However, there was a

significant decrease (p< 0.05) in the haemoglobin concentrations of group 6 (treated with

1:1v/v of the combined extract) when compared with group 1 (control) and groups 3 (treated

with 40ml/kg body weight of aqueous extract of M. oleifera extract) and 4(treated with

20ml/kg body weight of aqueous extract of T. occidentalis extract

Fig. 1 Effect of aqueous extracts of leaves of Moringa oleifera and Telfairia occidentalis

on the haemoglobin concentration (Hb conc.) of rats.

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves

Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

56

3.6 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the

Packed Cell Volume (PCV).

From figure 2, no significant (p>0.05) difference was observed in the packed cell volume of

the rats in the experimental groups when compared with the control during the initial seven

(7) days of treatment. However, after the 14th day of treatment, a significant (p< 0.05)

increase was observed only in the PCV of animals in group 4 when compared with those in

group 1. Group 4 (treated with 20ml/kg body weight of Telfairia occidentalis extract) was

able to increase the PCV of rats after 14th days of treatment but no significant (p> 0.05)

difference was observed between group 4 and the other experimental groups.

Fig. 2 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the

Packed Cell Volume (PCV).

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves

Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves

57

3.7 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on Red

Blood Cell (RBC) count.

From fig.3, the red blood cell counts of the animals showed no significant (p> 0.05)

difference in all the experimental groups after the first seven (7) days of treatment with the

plant extracts when compared with the control. But a significant (p< 0.05) increases was

observed in the red blood cell count of rats in groups 4 when compared with the group

1(control). A significant (p< 0.05) decrease was observed in group 6 when compared with

groups 4 and 3 after the 14th day of treatment.

Fig. 3: Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

Red Blood Cell (RBC) count.

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

58

3.8 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

Platelets count.

Figure 4 showed a significant (p< 0.05) decreases in the platelet counts of animals in groups 5

and 6 when compared with group 1 after the 7 days of treatment. Significant (p<0.05)

decrease was also observed in the platelet count of group 6 when compared to groups 2 and 3.

Significant (p<0.05) decrease was observed in the platelet counts of the animals in groups 6

and 5 when compared to those in groups 3 and 4 after the 14 days of treatment.

Fig 4 Effect of aqueous extracts of leaves of Moringa oleifera and Telfairia occidentalis

on Platelets count.

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves

Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

59

3.9 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on White

Blood Cell (WBC) count.

There was no significant (p> 0.05) difference in the white blood cell count of all the

experimental animals when compared with the control even after the 14th day of treatment as

showed in figure 5.

Fig. 5: Effect of aqueous extracts of leaves of Moringa oleifera and Telfairia occidentalis

on White Blood Cell (WBC) count.

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves

Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

60

3.10 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

Neutrophil count.

From figure 6, it was observed that group 4 (treated with 20ml/kg b.w of T.occidentalis)

showed a significant (p< 0.05) increase in the neutrophil count when compared with group 2

(treated with 20ml/kg b.w of M .oleifera) in the first seven (7) days of treatment. However,

when compared with the control (group 1), there was no significant difference in the

neutrophil count of all the animals in the treatment groups. Further treatment (Day 14) with

both extracts showed a significant (p< 0.05) increase in the neutrophil count of group 4 when

compared with group 1 and also with group 3.

Fig 6 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

Neutrophil count.

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1v/v)

61

3.11 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

Lymphocytes count.

From figure 7, it was observed that group 4 (treated with 20ml/kg b.w of T.occidentalis)

showed a significant (p< 0.05) increase in the lymphocytes count when compared with group

2 (treated with 20ml/kg b.w of M .oleifera) in the first seven (7) days of treatment. However,

when compared with the control (group 1), there was no significant difference in the

lymphocytes count of all the animals in the treatment groups. Further treatment (Day 14) with

both extracts showed a significant (p< 0.05) increase in the lymphocytes count of group 4

when compared with group 1 and also with group 3.

Fig. 7 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

Lymphocyte count.

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves

Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

62

3.12 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the

activity of Aspartate amino transferase (AST) of rats.

Figure 8 showed that there was no significant (p> 0.05) difference in the AST activity of all

the experimental animals treated with the aqueous extracts of the leaves of Moringa oleifera

and Telfairia occidentalis when compared with the control after the 14th

day of treatment.

Fig. 8 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the

activity of Aspartate amino transferase (AST) of rats.

Group 1 (control group) was given distilled water

Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

63

3.13 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the

activity of Alanine amino transferase (ALT) of rats

After day 14 of treatment, no significant (p>0.05) difference was observed in the ALT activity

of the treatment groups when compared to the control group. However, a significant (p<0.05)

increase was observed in the ALT activity of rats in groups 2 and 3 when compared to those

of group 5 as showed in fig. 9.

Fig. 9 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the

activity of Alanine amino transferase (ALT) of rats

Group 1 (control group) was given distilled water

Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves

Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

64

3.14 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on the

activity of Alkaline phosphatase (ALP) of rats From fig. 10, there was no significant (p<0.05) difference in the ALP activity of the test

groups when compared to those of the control group except in group 6 which showed a

significant (p<0.05) increase. A significant (p<0.05) increase was observed in the ALP

activity of rats in group 5 and group 6 when compared to group 1 after day 14 of treatment. A

significant (p<0.05) increase was also observed when the ALP activity of the rats in group 5

when compared to group 3.

Fig. 10 Effect of aqueous leaf extracts of Moringa oleifera and Telfairia occidentalis on

the activity of Alkaline phosphatase (ALP) of rats

Group 1 (control group) was given distilled water Group 2 was given 20ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 3 was given 40ml/kg b.w. of the aqueous extract of Moringa oleifera leaves Group 4 was given 20ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves

Group 5 was given 40ml/kg b.w. of the aqueous extract of Telfairia occidentalis leaves Group 6 was given 40ml/kg b.w of the aqueous extracts of T. occidentalis and M. oleifera leaves (1:1 v/v)

65

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 Discussion

Medicinal plants are of great importance to the health of individuals and communities and

their medicinal values lie in some chemical substances that produce definite physiological

actions on the human body (Edeoga et al., 2005).

The preliminary phytochemical analysis showed the presence of alkaloids, tannins, steroids,

flavanoids, fats and oils, carbohydrates, terpenoids, saponins, and proteins with the absence of

resins and reducing sugar in both leaves. However, glycoside was present in Moringa but

absent in fluted pumpkin. The quantitative phytochemical analysis indicated that leaves of

Moringa oleifera contain higher amounts of alkaloids and tannins than those of Telfairia

occidentalis. The alkaloid contents of Moringa oleifera and Telfairia occidentalis were 21.53

±0.53 mg/g and 12.15 ±0.42 mg/g and the tannin contents were 7.79 ± 0.05mg/g and 5.58 ±

0.02 mg/g respectively. However, the flavonoids and sterols content of leaves of Telfairia

occidentalis were found to be higher than those of Moringa oleifera. The flavonoids content

of Telfairia occidentalis and Moringa oleifera were 18.50 ± 0.14 mg/g and15.42 ± 0.10 mg/g

while their sterols values were 19.92 ±0.55 mg/g and 15.28±0.16 mg/g respectively. The most

important of these phytochemicals are alkaloids, tannins, flavonoids and phenolic compounds

(Hill, 1952). The high content of alkaloids in the leaves of these plants especially in Moringa

could be responsible for its use as a cardiac and circulatory drug (Makonnen et. al., 1997).

Most alkaloids have been showed to exert biological and pharmacological activities when

ingested by animals affecting the nervous system, particularly the action of neurotransmitters,

e.g. acetylcholine, adrenaline and dopamine (Schmeller and Wink, 1998). The results indicate

that the leaves of these plants posses some biologically active compounds which could serve

as potential sources of drugs.

The result of the acute toxicity (LD50) test of the aqueous leaf extracts of Moringa oleifera

and Telfairia occidentalis showed no toxicity on mice even when the concentration of both

extracts were increased to 5000 mg/kg body weight. Although the animals showed some signs

of weaknesses at 5000 mg/kg body weight, they became more active afterward. This is an

indication that the leaves of both plants are safe for human and livestock consumptions.

The result of the vitamin and mineral analysis showed that the aqueous extracts of the leaves

of Moringa oleifera and Telfairia occidentalis contain reasonable amounts of vitamin B6 and

66

folic acid and other mineral constituents necessary for erythropoiesis such as iron. Deficiency

of folic acid has been reported to cause macrocytic, megaloblastic and pernicious anaemia

(Rang et al, 2007). It has been shown that folic acid is effective in relieving the symptoms in

patients who have nutritional macrocytic anaemia, macrocytic anaemia of pellagra,

megaloblastic anaemia of pregnancy, and megaloblastic anaemia of infancy (Spies, 1962;

Chanarin et al., 2004). Iron is an essential constituent of the haem-moiety of haemoglobin,

thus deficiency of iron in humans and animals often leads to iron deficiency anaemia. These

hematinic agents may have contributed to the significant increase in the haematological

indices noticed when rats were fed aqueous leave extracts of Telfairia occidentalis (20ml/kg

body weight of extract) and Moringa oleifera (40ml/kg body weight of extract) within the two

weeks of treatment.

It was observed that there were no significant increase in the haemoglobin concentration of

rats in the experimental groups except in group 4 (treated with 20ml/kg b.w of Telfairia

oleifera) when compared with the control after the initial seven (7) days of treatment.

Telfairia oleifera has shown to contain substantial amounts of essential amino acids and iron.

The increase in the haemoglobin concentration could be attributed to the presence of these

amino acids and high iron content (1.38± 0.18 mg/g). Iron is an important component of

haemoglobin and functions in the transport of oxygen to cells and tissues. This was found to

be in line with the works of Agbede et al. (2008) who incorporated Telfairia occidentalis leaf

protein into infant weaning foods and observed a significant effect on the iron status. Alada

(2000) also noted that long term intake of diet supplemented with Telfairia occidentalis

significantly increased haemoglobin concentrations in rats. The increase may also be as a

result of enhanced iron bioavailability as recorded by (Hamlin and Latunde- Dada, 2011).

However, such significant increase was not observed in group 5 (treated with 40ml/kg body

weight of T. occidentalis extract). Nworgu (2007) found that broilers tolerated a lower level

of Telfairia occidentalis. When compared with group 2 (treated with 20ml/kg b.w of Moringa

oleifera extract), a significant (p< 0.05) increase was observed in the haemoglobin

concentration of animals in group 4. This is an indication that the aqueous extract of the

leaves of T. occidentalis could be a better haematinic than the Moringa oleifera aqueous

extract. This is in line with the work done by Olugbemi et al. (2010) where broiler chicken

were fed with Moringa oleifera leaf meal (MOLM) the result of their study showed no

significant increase in the haemoglobin concentration suggesting that MOLM does not

67

possess such blood tonic effects. The blood-building properties of Moringa leaves are not

well studied (Olugbemi et al., 2010). It has been reported that Moringa extract could produce

anaemic effect in rats when exposed for a long period of time (Adedapo et al., 2009). When

the effect of the combined extract on the haemoglobin concentration was compared with the

effect of the individual extracts, a significant (p< 0.05) decrease was observed in the

haemoglobin concentration. This effect may be attributable to the presence of isothiocyanate-

producing glycosides present in the aqueous extract of Moringa (Fahey, 2005). Glycosides

are ethers that link a sugar to a toxin called aglycone. Either the glycoside or the aglycone

alone may be toxic. The glycosides include cyanogenic glycosides. It is generally believed

that the toxic properties associated with cyanogenic glycosides are due to the hydrogen

cyanide (HCN) released from the glycosides by the activity of an enzyme complex. HCN has

a high affinity for iron and reacts with the trivalent iron of mitochondrial cytochrome

oxidase, the terminal respiratory enzyme linking oxygen with metabolic respiration (Adedapo

et al., 2009). Although this effect was not observed when the extracts were administered

alone, there could be a possibility of interaction of bioactive constituents in the polyherbal

formulation to produce an antagonistic effect.

The red blood cell counts of the animals in all the experimental groups showed no observable

increase in the red blood cell counts in the first seven (7) days of treatment with the plant

extracts. But further treatment after 14th day showed significant (p<0.05) differences in the

RBC counts of animals in group 4 when compared to the control. This shows that following

the administration of 20ml/kg body weight of Telfairia occidentalis and 40ml/kg body weight

of Moringa oleifera, the aqueous extracts of the leaves of these plants at these concentrations

could possibly stimulate erythropoietin release into the kidney. Erythropoietin, which is the

humoral regulator of RBC production increases the number of erythropoietin-sensitive

committed stem cells in the bone marrow that are converted to red blood cell and

subsequently to mature erythrocytes (Polenakovic and Sikole, 1996: SanchezElsner et al.,

2004; Ganong, 2005).

Interestingly, the administration of the combined extract produced a significant decrease in

the RBC counts after the 14th day of treatment when compared with groups 3 (40 ml/kg body

weight of Moringa extract) and 4 (20ml/kg body weight of T. occidentalis extract). The

reduction in the RBC count may be attributed to the increase in the iron content when the two

extract were combined. High concentration of free elemental iron has been implicated in lipid

68

peroxidation and as such could lead to the destruction of the red blood cell membrane and a

subsequent reduction in the red blood cell count. This could also be attributed to the

cumulative effect of tannins and saponins. Tannins at lower concentration are desirable for

humans and animal consumption (Liener, 1994). At high concentration, tannins have been

found to decrease the activity of digestive enzymes such as lipases and also reduce the

absorption of nutrients such as vitamin B12 (Doss et al., 2011). Vitamin B12 is an essential

vitamin in erythropioesis and its deficiency has been linked to pernicious and megaloblastic

anaemia. Saponins are known to posses lytic action on erythrocyte cell membrane. This

haemolytic action is due to their affinity for the membrane sterols particularly cholesterol with

which they form undissolvable complexes (Glauert et al., 1962). The reduction in RBC count

could mean that the extracts when combined may act antagonistically to lyse the membranes

of the RBCs, thus leading to their observed reduction.

The extracts showed no significant effects on the packed cell volume of the rats in the

experimental groups when compared with the control during the initial seven (7) days of

treatment. However, a significant increase was observed in the PCV of animals in group 4

when compared with those in group 1 after the day 14 of treatment. Group 4 (treated with

20ml/kg body weight of T. occidentalis) also showed an increase the PCV. Packed cell

volume (PCV) is a measure of the portion of the blood volume that is made up by red blood

cells. This is in line with the work of Oyedeji (2007) who reported an increase in PCV of two

severely anaemic paediatric patients when served T. occidentalis extract sweetened with milk.

Olaniyan and Adeleke (2005) served thirty anaemic pregnant women from rural communities

with an oral mixture of T. occidentalis leaves, milk and raw egg three times a day for seven

days and observed a significant rise in mean packed cell volume (PCV). The increase in the

PCV could be as a result of the increase in the red blood cells observed in this group. The

non-significant effect of the extract of Moringa oleifera at 20 and 40ml/kg body weight on the

PCV throughout the experimental period is an indication that there was no destruction of

matured cells and no change in the rate of blood cells production (haematopoiesis). This

further show that the aqueous leaf extract of Moringa oleifera has little potential to stimulate

haematopoiesis.

A significant decrease was observed in the platelet counts of the animals in group 6 when

compared to those in groups 2, 3, 4, 5. The decrease was time and dose dependent. This

shows that the extracts had a negative effect on the platelet counts of the animals. This could

69

be as a result of the inhibition of the release of thrombopoietin, a regulator of thrombopoiesis

or as a result of the inhibition of vitamin k, which is an important factor in the blood

coagulation process. Reduction in platelet count could reduce the ability of the blood to clot

and could lead to death from excessive bleeding.

The result of the effect of white blood cell counts of rats treated with aqueous extract of

Moringa oleifera and Telfairia occidentalis showed no significant difference in all

experimental groups when compared with the control during the period of the experiment.

The non significant effect on the WBC may imply that there was no change in the body’s

ability to respond to infection. This may also imply that both extract do not pose any

challenge on the immune system.

The result of the effect of aqueous extracts of the leaves of Moringa oleifera and Telfairia

occidentalis on the neutrophils and lymphocytes of rats showed a significant increase between

groups 4 when compared to group 2 during the first seven (7) days of treatment. A significant

increase was also observed in the neutrophils and lymphocyte of rats in group 4 when

compared to the control and group 3 after 14 days of treatment. The percentage increase in the

neutrophils and lymphocytes of animals in group 4 may be an increased ability of the

neutrophils to phagocytosize (cellular ingestion of offending agents) (Dacie and Lewis, 2001).

Lymphocytes are the main effectors cells of the immune system (Mcknight et al., 1999). The

administration of Telfairia occidentalis extract appears to exhibit stimulatory effect on the

effectors cells of the immune system.

The result of the effect of the aqueous extracts of the leaves of Moringa oleifera and Telfairia

occidentalis on the liver marker enzymes showed no significant increase in the AST

activities. The effects of the extracts on the ALP activity showed no significant increase in

the experimental groups except groups 5 and 6 that showed a significant increase in the ALP

activity. Alkaline phoshpatase, ALP, is a plasma and endoplasmic reticulum membrane-

bound enzyme (Wright et al., 1972). It is not specific to the liver and an increase could also

be seen during period of active bone formation. So an increase in its activity as observed in

groups 5 and 6 does not necessarily indicate hepatotoxicity.

When compared to the control, no significant difference was observed in the ALT activity of

rats in all experimental groups. Significant increase was observed in the ALT activity of the

rats in groups 2 and 3 (20ml/kg and 40ml/kg body weight of Moringa oleifera extract

70

respectively) when compared with the group 5 (40ml/kg body weight of Telfairia

occidentalis extract). Serum ALT is a more specific marker of liver damage (Plaa and Hewitt,

1982). High levels of this enzyme in the serum may indicate liver damage, muscle injury and

hepatic necrosis (Roper, 1987). Therefore an increase in the activity of this enzyme observed

when rats were treated 40ml/kg body weight of Moringa oleifera extract probably indicate

that prolonged and excessive use may be hepatotoxic. This is in line with the work of

Oluduro and Aderiye (2009) that showed that prolonged consumption of water treated with

concentrations greater than 2 mg /ml of M. oleifera seed extract constitute liver infarction.

However, at low concentration of the extracts, no increases in the activity of these enzymes

were observed. This shows that at low concentration, the extracts are safe for consumption

and pose no threats to the liver cells but as the concentration increased, signs of toxicity may

be observed.

4.2 Conclusion

The results of this study indicate that the aqueous extracts of the leaves of Telfairia

occidentalis and Moringa oleifera at 20ml/kg body weight and 40ml/kg body weight

respectively may possibly serve as an acceptable blood booster in an anaemic condition or

used for prophylactic purposes without any significant toxic effects on the liver in rats.

Although the specific mechanism(s) through which the extracts enhance blood volume was

not ascertained in this study, it is suggested that the extracts may have a direct effect on the

body system that produces blood cells and contains constituent(s) that can interact and

stimulate the formation and secretion of erythropoietin, hematopoietic growth

factors/committed stem cells. This suggests that the aqueous extracts of the leaves of these

plants possesses haematinic properties. This haematopoietic effect may be due to the high

content of different minerals in the leaves of these plants. Although the aqueous extracts could

be helpful substitutes in cases of blood shortage or other conditions which places high

demand on the blood forming system of the mammalian body such as pregnancy, caution

should be exercised when using Moringa oleifera as prolonged use at concentration of

40ml/kg body weigh could increase the activity of ALT in the serum. From the results of the

study, it could be seen that Telfairia occidentalis is a better haematinic agent and does not

pose a threat to the liver. It was also observed that when combined, the extracts produced an

antagonist effect as shown in the decrease in RBCs, HB and an increase in the ALP activity.

71

4.3 Suggestions for further studies

The effects of aqueous extracts of the leaves of Moringa oleifera, Telfairia occidentalis and

their combined extract on normal rats was investigated in this study and it is therefore

suggested that further studies be done on

• The effect of aqueous extracts of the leaves of Moringa oleifera, Telfairia

occidentalis and their combined extract on anaemic rats to ascertain the effect on

anaemic rats.

• To identify the actual compound(s) responsible for the antagonistic effect observed

when the extracts were combined.

• The plant extract posed no toxic effects on the liver during the short period of

treatment, prolonged treatment of the plant extract is then suggested. This is to

ascertain its prolonged effect on the liver of rats.

72

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Appendix I

Table 2: Absorbance and activity of ALT in the serum

Absorbance U/L Absorbance U/L

0.03 4 0.28 48

0.05 8 0.30 52

0.08 12 0.33 57

0.10 17 0.35 62

0.13 21 0.38 67

0.15 25 0.40 72

0.18 29 0.43 77

0.20 34 0.45 83

0.23 39 0.48 88

0.25 43 0.50 94

Table 3: Absorbance and activity of AST in the serum

Absorbance U/L Absorbance U/L

0.02 7 0.12 47

0.03 10 0.13 52

0.04 13 0.14 59

0.05 16 0.15 67

0.06 19 0.16 76

0.07 23 0.17 89

0.08 27

0.09 31

0.10 36

0.11 51

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Appendix II

Table 6: Phase I of the acute toxicity (LD50) of the aqueous extracts of the leaves of

Moringa oleifera and Telfairia occidentalis

Dosage (mg/kg bodyweight)

Mortality in group given Moringa oleifera

Mortality in group given Telfairia occidentalis

Group 1 10 0/3 0/3

Group 2 100 0/3 0/3

Group 3 1000 0/3 0/3

Table 7: Phase II of the acute toxicity (LD50) of the aqueous extracts of the leaves of

Moringa oleifera and Telfairia occidentalis

Dosage (mg/kg bodyweight)

Mortality in group given Moringa oleifera

Mortality in group given Telfairia occidentalis

Group 1 1600 0/3 0/3

Group 2 2900 0/3 0/3

Group 3 5000 0/3 and ST 0/3 and ST

ST= Signs of Toxicity.