BIOAVAILABILITY OF IRON FROM BASELLA ALBA AND AMARANTHUS HYBRIDUS LEAVES SUPPLEMENTED DIET IN IRON DEFICIENT ANAEMIC ALBINO RATS.
BY
Ceaser Antiya, MOSES
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCE
AHMADU BELLO UNIVERSITY
ZARIA
JANUARY, 2016
i
BIOAVAILABILITY OF IRON FROM BASELLA ALBA AND AMARANTHUS HYBRIDUS LEAVES SUPPLEMENTED DIET IN IRON DEFICIENT ANAEMIC ALBINO RATS.
BY
Ceaser Antiya MOSES, B.SC BIOCHEMISTRY (A.B.U) 2011 MSc/SCIE/44812/2012-2013
A DISSERTATION SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTERS DEGREE IN BIOCHEMISTRY
DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCE AHMADU BELLO UNIVERSITY, ZARIA NIGERIA
JANUARY, 2016
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DECLARATION
I hereby declare that the work in this dissertation entitled ―BIOAVAILABILITY OF IRON FROM BASELLA ALBA AND AMARANTHUS HYBRIDUS LEAVES SUPPLEMENTED DIET IN IRON DEFICIENT ANAEMIC ALBINO RATS” has been performed by me in the Department of Biochemistry under the supervision of Mr. O. A. Owolabi and Prof. E. Oyinke. The information herein derived from literature has been duly acknowledged in the text and a list of references provided. No part of this dissertationwas previously presented for another degree or diploma at any university to the best of my knowledge.
…………………………………. ……………………… …………………… Moses Ceaser Antiya Signature Date
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CERTIFICATION
This dissertation entitled ―BIOAVAILABILITY OF IRON FROM BASELLA ALBA AND AMARANTHUS HYBRIDUS LEAVES SUPPLEMENTED DIET IN IRON DEFICIENT ANAEMIC ALBINO RATS” by Ceaser, Antiya MOSES meets the regulations governing the award of the degree of MASTER of Science Ahmadu Bello University, and is approved for its contribution to knowledge and literary presentation.
Mr. O. A. Owolabi ………………………. ……………………………… Chairman, Supervisory Committee Signature Date
Prof. E. Onyike ………………………. ……………………………… Member, Supervisory Committee Signature Date
Prof. M. N. Shuaibu ……………………….. ..…………………………....Head of Department Signature Date
Prof. K. Bala ……………………….. ……………………………… Dean, School of Postgraduate Studies Signature Date
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DEDICATION This work is dedicated to my late Father, Mr. Moses Antiya
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ACKNOWLEDGEMENT
I must first of all express my gratitude to God for his unfailing love, grace and mercy to me; I give him Glory for his faithfulness, provision, and divine health he has granted me throughout my period of study in Zaria.
My profound gratitude to my project supervisors, Mr. O. AOwolabi and Prof. E. Onyike for their limitless effort and suggestions, constructive criticism, corrections, close supervision and inspiration during the course of this work. It was your assistance, in all ramifications that made this work a success.
My deep appreciation goes to my Head of Department, Prof. I.A Umar and the entire academic and nonacademic staff of the Department of Biochemistry for their individual and collective support.
I will like to specially appreciate Dr. D. B James for her guidance and encouragement during the course of my studies. I will also like to acknowledge the staff of animal house Department of Pharmacology, Faculty of Pharmaceutical Science for allowing me to use the animal house for the course of this study.
I sincerely want to appreciate my dear friends OkoloIjeoma, BindaAndongma, Ugoji Cynthia, Daniel Abba, AyubaDanburam, Ann Chinedu, Femi Omogoye, Mike Drenkat, KambaBayo, Samson Melah, Sale Gelte, Gideon Rimamfate, Josiah Angut, IfeomaUche, Ebuka Samuel,Chidi Anthony, Atobiloye Ahmed andAbdulmuttalab Baba-Ali. You people made the project experience a pleasant one.
Finally, I am grateful to the entire Moses Antiya family for your prayers, emotional and financial supports that made my studies a huge success.
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ABSTRACT
Iron deficiency anaemia is the most common form of micronutrient deficiency especially in the developing countries.The bioavailability of iron from blanched and unblanched Basella alba and
Amaranthus hybridus leaves supplemented diet in iron deficient anaemic albino rats was investigated by determining vitamin C, antinutrients, speciation pattern of iron and effect of the leaves supplemented diet on serum ferritin and some haematological parameters using standard methods. Forty eight young albino rats were divided into eight groups. After induction of anaemia by feeding with iron deficient diet and deionized water, diet supplementation was carried out for 21 days. Amaranthus hybridus has a significantly (p<0.05) higher content of vitamin C (35 mg/100g), phytate (2.06 g/100g) and oxalate (89 mg/100g) than Basella alba.
Basella alba recorded a significantly (p<0.05) higher levels of Fe, Zn, Cu, Ca and Mg than
Amaranthus hybridus. Blanching significantly (p<0.05) decreased the vitamin C, phytate, oxalate and mineral content of both Basella alba and Amaranthus hybridus. Iron speciation pattern in the leaves showed that exchangeable and the residual forms of iron predominates over reducible and exchangeable in both leaves, which increased in B.alba but decreased in A.hybridus after blanching.After diet supplementation for 21 days, diet supplemented groups recorded significant
(p<0.05) increase when compared with the anaemic rats. Groups supplemented with 10% unblanched A. hybridus recorded a significant increase in serum ferritin (138.40 ng/ml), red blood cell count (RBC) (11.97 x 106µl) and packed cell volume (PCV) (40.67%) when compared to the anaemic rats. Similarly, diet supplementation with 10% unblanched and blanched Basella alba and Amaranthus hybridus leaves respectively significantly (p<0.05) increased hemoglobin
vii concentration, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration but recorded no significant (p>0.05) increase in the mean corpuscular volume when compared with the anaemic rats. The results suggest that Basella alba and Amaranthus hybridus supplemented diet increase bioavailability of iron in iron deficient anaemic albino rats with unblanched vegetables exhibiting greater potentials than the blanched.
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TABLE OF CONTENTS
Page
Cover Page ………………...…………..……………………………………………………….i
Fly Page ……………………………………………………………………….………..…..ii
Title Page ………………………………………………………….………….……………..iii
Declaration Page ……………………………………………………………………………….....iv
Certification Page ………..…………………………………………..…………………………….v
Dedication ……….………….……………………………………...……………………………..vi
Acknowledgment .……………………………………………...………………………...... vii
Abstract ..………….……………………………………………..……………………….viii
Table of Contents …….…………………..………………………………………………………..x
List of Tables …....………………………...…………………………………………………..xvii
List of Figures ……...……………………....…………………………………………………..xviii
List of Plates …….…………………………..………………………………………………..…xix
List of Appendix ………………………..……………………………………………………....xx
List of Abbreviation ………………………….………………………………………………....xxii
CHAPTER ONE
1.0 INTRODUCTION ………………….....…………………...………...…………………...1
1.1 Statement of Research Problem ……..….………………………………...... 3
1.2 Justification …..……….……………..………………………….….………...... 4
1.3 Aim and Objectives …………………..…………………..………..…………………..…5 ix
1.3.1 Aim .……………………………………………………………...……………………..5
1.3.2 Specific Objectives .…….………………………..……….…………….……………....5
CHAPTER TWO
2.0 LITERATURE REVIEW ….…………………………….……………………………….6
2.1 Basella alba and Amaranthus hybridus ...………..…………………………………….…6
2.1.1 Origin and distribution ……………………………………….………...……………….…6
2.1.2 General description ………………………………………………………………………..7
2.1.3 Botanical information ……………………………………………………………..9
2.1.4 Classification …..…………..……..………………….…………………………………...10
2.1.5 Chemical composition …………………...…………………………………………...….11
2.1.6 Uses …………………………………………………………..…………………………..12
2.1.6.1 Culinary uses ...…………………...……………………………………………..12
2.1.6.2 Medicinal uses ………..…………………………..…………...………………………...13
2.1.6.3 Industrial uses ………...………………………………………………………………....14
2.2 Iron ………………………………………………………...……...…………………….15
2.2.1 Occurrence ………………………………………………………………………………15
2.2.2 Iron requirement and distribution in the body …………...…..……………...... 15
2.2.3 Dietary iron ………………………….………………………..……………………….16
2.2.4 Iron absorption …………………………………………..…………...... 17
2.2.4.1 Non-haem iron absorption …...……………………………...…..………...... 18
2.2.4.2 Haem iron absorption……………...……………..……..………………………...……..19
2.2.5 Iron Deficiency …………...………………….……………………………………20
x
2.2.6 Major causes of iron deficiency anaemia …………….…..…...……………………..21
2.2.6.1 Blood Loss ……………………………………………………………………………21
2.2.6.2 The Maternal– Fetal Bridge of Iron Deficiency ……………………………………21
2.2.6.3 Malaria ………………..………………………………………………...... 23
2.2.6.4 Hookworm ……..…….……………………………………………………….………23
2.2.7 Some haematological parameters and their role in diagnosis of iron
deficiency anaemia …...………………………………………………………………24
2.2.7.1 Haemoglobin Concentration …...……………………..…………..……………………..24
2.2.7.2 Serum ferritin concentration ……...…..……...……………………..……………………25
2.2.7.3 Total Iron Binding Capacity and Transferrin Saturation ………….……………………27
2.2.7.4 Serum Transferrin Receptor ……….…...………..………………...... …………………..27
2.2.7.5 Serum Iron Concentration …..…….…………………………………………………….28
2.2.8 Prevention of Iron Deficiency ……….………………………………...... ………………28
2.2.8.1 Dietary Diversification ..……………..…...…………………………….……………….28
2.2.8.2 Food Fortification …….……………………..……………………………………….28
2.2.8.3 Supplementation …………………………………………...... …………………….29
2.3 Blanching …………..……..……………………………………………..……………....30
2.4Antinutrients ………………………………………………………………………..…30
2.4.1 Antinutrtional factors…………………………………………………………………….30
xi
2.4.2 Biochemical effects of some anti-nutritional factors……………………………………..31
2.4.2.1 Phytate ...... 31
2.4.2.2 Oxalate
……………………………………………………………………………312.4.2.3
Saponins ……………………………………………………………………………32
2.4.2.4 Tannins ……………………………………………………………………………32
2.4.2.5 Cyanogenic glycosides ……………………………………………………………33
2.4.2.6 Lectins ……………………………………………………………………………………33
2.4.2.7 Protease inhibitors ……………………………………………………………………33
CHAPTER THREE
3.0 MATERIALS AND METHODS ………………………..………...….………………35
3.1 Materials …….……………………………………………………..……………...... 35
3.1.1 Chemicals ….…………………………………………………………………..…….35
3.1.2 Collection of plant materials ……..………………..……………..………..……………..35
3.1.3 Animals ..………………………………….……………………...…………………..….35
3.2 Methods ……...……………………………………...………………………………..…36
3.2.1 Preparation of vegetables leaves …………..…………….…….….……………………..36
3.2.2 Blanching of the vegetables ………………….…….…………...………………………...36
3.2.3 Quantitative determination of vitamin C …...……...... ………………………………36
3.2.4 Quantitative determination of some antinutrients ………………………………....36
3.2.4.1 Phytic acid determination …..……………………………………..…………………..36
3.2.4.2 Oxalic acid determination …..………………………….…………………………….…37
3.2.5 Quantitative determination of mineral content ..……..……………………………38 xii
3.2.5.1 Sample digestion ………...…………………………..……………….………………....38
3.2.6 Determination of the speciation pattern of iron. ...…………...……………………..39
3.2.6.1 Exchangeable phase…………………………...... ……………………………………39
3.2.6.2 Carbonate phase (exchangeable phase)…….………………………………………..….39
3.2.6.3 Amorphous Fe oxyhydroxide phase (reducible) ….....……………………………….39
3.2.6.4 Crystalline Fe oxide phase (reducible) …….……………...…………..…………...... 39
3.2.6.5 Sulphides and organic phase (oxidizable) …...…………...…………………………..40
3.2.6.6 Silicates and residual phase ...……………………...………………………..………….40
3.2.6.7 Invitro bioavailable iron …………….……………………………...... 40
3.2.7 Diet formulation ….…………………….………...…………………………………….41
3.2.8 Induction of anaemia ………..………………..…….……………………..……………41
3.2.9 Animal grouping …………………………………..……...……...…………………….42
3.2.10 Collection of blood sample …………………………………………………………42
3.2.11Determination of haematological parameters level ……….....…...…...………………...43
3.2.11.1 Packed cell volume ………………….....………………………..……....……………....43
3.2.11.2 Haemoglobin concentration …….….……………………………………..………..…..43
3.2.11.3 Red blood cell and white blood cell count …………….……………..…………..……...45
3.2.11.4 Determination of mean corpuscular volume, mean corpuscular
haemoglobin and mean corpuscular haemoglobin concentration
..………..………..……...….45
3.2.12Determination of serum ferritin concentration …..………………………………..46
3.3 Statistical Analysis………………………………………………..……..………………..47
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CHAPTER FOUR
4.0 RESULTS ….……………...…………………………………………………………48
4.1 Vitamin C Content of Blanched and Unblanched Basella alba and
Amaranthus hybridus Leaves ………………………..………………………..…48
4.2 Antinutrients Content of Blanched and Unblanched Basella alba
andAmaranthus hybridus Leaves ….…………….…………………………………..48
4.3 Mineral Content of Blanched and Unblanched Basella alba and
Amaranthus hybridus Leaves ………………………………………………...….52
4.4 Speciation Pattern of Iron in Blanched and Unblanched Basella alba
andAmaranthus hybridus Leaves ...... 52
4.5 Weight Change of Iron Deficient Rats Supplemented With
Blanched and Unblanched Basella albaand Amaranthus hybridus Leaves …....55
4.6 Effect of Diet Supplemented with Blanched and Unblanched
Basella albaand Amaranthus hybridus leaves on
Serum Ferritin Concentration …………………………………………………....55
4.7 Haemoglobin Iron Concentration of Iron Deficient Rats Supplemented
with blanched and Unblanched Basella albaand
Amaranthus hybridus Leaves …………………………………………………………....55
4.8 Effect of Blanched and Unblanched Basella albaand Amaranthus
hybriduson Some Haematological Parameters ….…………………………..…….60
CHAPTER FIVE
5.0 DISCUSSION …….……………………………………………………………………...62
xiv
CHAPTER SIX
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS ……...…………….68
6.1 Summary ...... …………………………………………………………………..……...68
6.2 Conclusion …………………...………………………………………………………….69
6.3 Recommendation ………….………………………………………………………...69
References ……...……………………………………………………………………………...71
Appendix ……….…...………………………………………………………………………89
xv
LIST OF TABLES
3.1 Composition of iron deficient and iron sufficient diet………………..…..……...………41
4.1 Some Mineral Content of Blanched and Unblanched Basella
albaand Amaranthus hybridus Leaves …………………..…………………..……53
4.2 Iron Speciation Pattern of Blanched and Unblanched
Basella albaand Amaranthus hybridus Leaves ...... 54
4.3 Effect of Blanched and Unblanched Basella alba and
Amaranthus hybridus Leaves Supplemented Diet on Some Haematological
Parameters in Iron Deficient Rats …………………………………...……………….61
xvi
LIST FIGURES
4.1 Vitamin C Content of Unblanched and Blanched Basella alba and
Amaranthus hybridus Leaves ………………………………...………………….49
4.3 Phytate Content of Blanched and Unblanched Basella albaand
Amaranthus hybridus Leaves ……………………………………………………50
4.3 Oxalate Content of Blanched and Unblanched Basella alba and
Amaranthus hybridus Leaves ………………………………………………....…51
4.3 Effect of Blanched and Unblanched Basella albaand
Amaranthus hybridus Leaves Supplemented Diet on Weight Change
of Iron Deficient Rat ……………………………….…………………………………...57
4.4 Effect of Blanched and Unblanched Basella alba and
Amaranthus hybridus Supplemented Diet on Serum Ferritin of Iron
Deficient Anaemic Rats ……………………………………...……………...... 58
4.5 Effect of Blanched and Unblanched Basella alba and
Amaranthus hybridus Leaves Supplemented Diet on Haemoglobin
Concentration of Iron Deficient Rats ……………………………………………………59
xvii
PLATES
Page
Plate 2.1The Photograph of Basella alba and Amaranthus hybridus leaves ………………………..…….8
xviii
LIST OF APPENDICES
Page
Appendix 1 Experimental Design ………………...…………………………………………89
Appendix 2 Content Vitamin and Mineral mix ……...………………..……………………..90
Appendix 3 Standard Curve for Vitamin C ….…………………………………………..91
Appendix 4 Standard Curve for Fe ………...…………………………………….. ……92
Appendix 5 Standard Curve for Zn …….…………………………………………….....93
Appendix 6 Standard Curve for Cu …….…………………………………………….....94
Appendix 7 Standard Curve for Mg ……..………………………………………………95
Appendix 8 Standard Curve for Ca ….………………………………………………….96
Appendix 9 Standard Curve for Serum Ferritin ……..…………………………………….97
Appendix 10 Vitamin C content in Blanched and Unblanched Basella alba
andAmaranthus hybridus Leaves ……………………………………………98
Appendix 11 Antinutrients Content of Unblanched and Blanched Basella alba
andAmaranthus hybridus Leaves ……………………………………………99
Appendix 12Effect of Blanched and Unblanched Basella alba and
Amaranthus hybridus Leaves Supplemented Diet on Weight Change of
Iron Deficient Rat ………………………………..…………………………..100
Appendix 13Effect of Blanched and Unblanched Basella alba and
Amaranthus hybridus Leaves Supplemented Diet on Haemoglobin
Concentration of Iron Deficient Rats ……………………………………101
xix
Appendix 14 Effect of Basella alba and Amaranthus hybridus Leaves Supplementation
on Serum Ferritin Concentration
of Iron Deficient Rats ……………………………………………………………102
Appendix 15 Preparation of Reagents Used for Speciation Pattern Analysis ……………103
Appendix 16Preparation of Stock solution of standard drug ……………………………104
Appendix 17 Mineral content of control and supplemented diets …………………...…………105
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LIST OF ABBREVIATIONS
%: Percentage
A hybridus Amaranthus hybridus
B. alba Basella alba
Ca Calcium
Cd Cadmium
Cu Copper
DMT Divalent Metal Transporter
FAO Food and Agriculture Organization
Fe: Iron
FLVCR Feline Leukemia Virus subgroup C Receptor-related protein
H Hydrogen
Hb Haemoglobin
HCP Human Carrier Protein hDMT Human Divalent Metal Transporter
IRP Iron Regulatory Protein
IRE Iron Response Element
xxi
MCH Mean Corpuscular Haemoglobin
MCHC Mean Corpuscular Haemoglobin Concentration
MCV: Mean Corpuscular Volume
Mg Magnesium
Mn Manganese
MTF Metal Transcription Factor
Na Sodium
PCV Packed Cell Volume
PCFT Proton-Coupled Folate Transporter
RBCs Red Blood Cells
STfR Soluble transferrin receptor
Tf Transferrin
TIBC Total Iron Binding Capacity
UNICEF United Nation Children Fund
WBCs White Blood Cells
WHO World Health Organization
Zn Zinc
xxii
CHAPTER ONE
1.0 INTRODUCTION
Anaemia is defined as a pathological process characterized by abnormally low haemoglobin (Hb) concentration in red blood cells, less than 13g/dl in male and
12g/dl in females (Okochi et al., 2003). In developing countries, anaemia is a major contributory factor to maternal morbidity and mortality (Saeed, 1996). Malaria,
HIV/AIDS, hookworm infestation, schistosomiasis, and other infections such as tuberculosis are particularly important factors contributing to the high prevalence of anaemia in some areas (Adusi-Poku et al., 2008; WHO 2014). In the tropics, due to endemicity of malaria, between 10 to 20% of the population presents less than
10g/dl of Hb (Diallo et al., 2008). However research has revealed that iron deficiency is the cause of most forms of anaemia (Viteri, 1993).
Iron deficiency is the single mostcommon nutritional deficiency and the major causeof anaemia, affecting more than 2 billion people worldwide. It has a substantial effect on the lives of young children, women, postpartum and premenopausal women due to high irondemand during infant growth, pregnancy and loss of iron during menstruation. Iron deficiency anaemia can result from inadequate iron intake, decreased iron absorption, increased iron demand and increased iron loss (Stoltzfuset al., 2004; WHO, 2008;
McLean et al., 2009).
1
Iron deficiency anaemia is characterized by the reduction or absence of iron (Fe) stores, low serum concentration of Fe, poor Hb concentration, haematocrit reduction and increased platelet count. Iron deficiency anaemia is particularly prevalent in developing countries (Viteri, 1993) and due to its effects on development, growth, resistance to infections and association with the mortality of infants younger than 24 months, it is considered the major public health problem (McLean et al., 2009; Ghada et al., 2010).
Iron is vital for oxygen transport and energy metabolism (De Rosa, 2007).Its deficiency or overload disturbs the biochemical and physiological balance of the body. The consequences of iron deficiency anaemia include impaired growth, retarded psychomotor and cognitive development, damaged immune mechanisms with increased morbidity and mortality rates (WHO, 2001; Neumann and Bwibo, 2004).
Diet offers a greater and more diverse group of plant bioactive substances which help to protect the body from disease and regulate the body process on which vitality and good health depend.Therefore vegetables in diet play an important part in maintaining general good health owing to the presence of nutritional and phytochemical property. (Anon et al., 2005; FAO, 2005).
In nature, there are many vegetables of high nutritive value, which can be a good source of micronutrients. They have however remained underutilized due to lack of awareness and popularization of technologies for utilization (Sheela, 2004).Vegetables
2 also contain an immense variety of bioactive compounds with health promoting factors such as antioxidants,phytochemicals, essential fatty acids and dietary fiber (Gupta et al.,
2008).
Basella albais a highly succulent vegetable similar to waterleaf (Harry, 2000).Of many spinach cultivars; Indian spinach is the most cultivated in Africa which is probably due to good environmental factors such as high temperature, high rainfall and moist fertile soils with high organic matter observed in the African continent (Procheret al., 1995).Basella alba is used largely as food in the western part of Nigeria.It also serves as raw materials for the production of many useful products. Basella alba has been found to be an important source of some vitamins and also useful in the treatment of many health related diseases (Haskell et al., 2004; Rahmatullah et al., 2010; Ramu et al., 2011).
Amaranthus species are a highly popular group of vegetables that belong to different species, it is known as ‗inene‘ in south eastern Nigeria and ‗alaihu‘ in northern
Nigeria. Amaranthus hybridus, commonly called smooth amaranth, smooth pigweed, red amaranth, or slim amaranth it is a species of annual flowering plant whichcan grow up to
2m.Amaranthus hybridus is widely grown in West Africa, Indonesia and Malaysia
(Hugues and Phillip, 1989).
Amaranthus is highly nutritious, both the grain amaranth and leaves are utilized for human as well as for animal food (Tucker, 1986). The plant has been used in treating
3 dysentery, ulcers and haemorrhageof the bowel due to its astringent property (Krochmal and Krochmal,1973). Leaves possess antibacterial effect (Cyrus et al., 2008), cleansing effect and also help to reduce tissue swelling (Bhattacharjee, 2004).
1.1 Statement of Research Problem
Iron deficiency is the most common and widespread nutritional disorder in the world
(Stoltzfus et al., 2004), affecting children and women in the developing countries. Iron deficiency affects more people than any other disease condition, constituting a public health condition of epidemic proportion.It‘s also the only nutritional deficiency that is significantly prevalent in industrialized countries (WHO, 2014). Iron deficiency anaemia has high prevalence especially in the developing countries.According to National Food
Consumption and Survey in 2003, 43.7% of pregnant women, 24% of women and 34% of under-five children suffer from iron deficiency anaemia in Nigeria(Maziya-Dixonet al,. 2004).
In developing countries such as Nigeria,blood loss, pregnancy, infections such as malaria, hookworm infestation, tuberculosis, schistosomiasis and malnutrition associated with reduced dietary intake of iron containing diet are factors contributing to the high prevalence of iron deficiency anaemia (Burgmann et al., 1996; Bungiro and Cappello,
2010; Erhabor et al., 2013; Ekwochi et al., 2014).
4
Iron deficiency anaemia can result in impaired cognitive development, reduced physical work capacity of an individual or population which have serious economic consequences and slows national development. In severe cases, iron deficiency anaemia increased risk of mortality, particularly during the perinatal period. (WHO, 2001). Iron deficiency also affect all body functions, not only through anaemia which appears late in the process of tissue iron deficits (Ghosh et al., 1995).
1.2 Justification
Iron is very essential to the body, it functions primarily as a carrier of oxygen in the body both as a part of haemoglobin in the blood and myoglobin in the muscle. Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, which include; DNA synthesis and electron transport. Ironalso has a critical role within cells assisting in oxygen utilization, enzymatic systems, especiallyfor neural development, and overall cell function in the body(Ghosh et al., 1995).
Traditionally diet supplementation has been practiced to ensure the adequacy of the diet, they add essential nutrient to diet so as to overt deficiency diseases and prevent adverse effect of marginal nutrient inadequacy (Webb, 2011). Some vegetables are found to contain iron, a well-balanced vegetarian or vegan diet provides plenty of iron which can contain as much or more iron than mixed diets containing meat (Hunt, 2003; Harvey et al., 2005). Therefore this research is aimed at determining the bioavailability of iron from
Basella alba and Amaranthus hybridus leaves supplemented dietin iron deficient rats.
5
1.3 Aim and Objectives.
1.3.1 Aim
To evaluate the in-vitro and in-vivo bioavailability of iron from Basella alba and
Amaranthus hybridusleaves supplemented diet in iron deficiency anaemic albino rats.
1.3.2 Specific objectives
The specific objectives are to determine;
I. The vitamin C, phytate and oxalate content of the leaves.
II. Some mineral (Iron, Calcium, Magnesium, Zinc and Cupper) content of
the leaves.
III. The speciation pattern; in-vitrobioavailability of iron content of the leaves.
IV. The effect of Basella alba and Amaranthus hybridus leaves supplemented
diet on the weight change, serum ferritin and some haematological
parameters of iron deficient anaemic albino rats
CHAPTER TWO
2.0 LITERATURE REVIEW
6
2.1 Basella alba andAmaranthus hybridus
2.1.1 Origin and distribution
Basella is native to tropical Southern Asia, probably originated from India or Indonesia
(Saroj et al., 2012). Basella alba is particularly abundant in Malaysia, Philippines, tropical Africa, Caribbean and tropical South America (Palada and Crossman, 1999),
Southeast of Brazil (Echo Plant Information Sheet, 2006). Due to easy adaptation to a variety of soils and climates,Basella alba is considered one of the best tropical spinach throughout the tropical world (Palada and Crossman,1999).
Amaranthus hybridus originated as riverbank colonizers in eastern North America, with earlier range expanding throughout milder and moister regions of Mexico, Central
America, and Northern South America. The earliest European records of the species date back approximately 300 years, with spread in Europe taking place primarily in the
Mediterranean region (Trucco and Tranel, 2011). Spread of Amaranthus hybridus has been slower than that of other Amaranthus weeds, especially when compared to
Amaranthus retroflexus. Presence of the species in western North America, eastern Asia,
Australia, and South Africa has been reported as of early to mid-1900s. Today
Amaranthus hybridus is a globally distributed weed of agricultural fields and it ranks among the 18 most serious weeds in the world (Holm et al.,1991).
7
2.1.2 General description of the plants
Basella is a highly succulent leafy vegetable that belongs to the family Basellacea.It is commonly known in English as Indian spinach, Ceylon spinach or Malabar spinach and locally called ―Amunu-tutu‖ in Southwestern Nigeria. It‘s a fast growing perennial plant with a succulent, branched, smooth, twining herbaceous vine that grows several meters
(10metres or 33fts) in length. It‘s also a valuable vegetable that can be cultivated from either seed or by cutting (Adhikari et al., 2012).The stems are purplish or green and are quadrangular in shape.They are about 1 to 3 cm thick, with prominent nodes and antinodes.The leaves are fleshy, ovate or heart-shaped, chordate base, dark green in color, glossy above and glaucous below 5 to 12 cm long, stalked, tapering to a pointed tip.The spikes are axillary, solitary, 5-29 cm long. The flowers are inconspicuous, bisexual white flowers borne on axillary spikes or branching peduncles. Fruit is fleshy, stalk less, ovoid or spherical, 5-6 mm long, and purple when mature (Kumaret al., 2013a).
Amaranthus hybridus is a basal specie of the crop subgenus.It is commonly called
―Amaranth or pigweed‖.It is also known as spinach in English, ‗Efo‘ in South Western
Nigeria, ‗Inene‘ in South Eastern Nigeria and ‗Alaiyahu‘ in Northern
Nigeria.Amaranthus hybridus is an annual herbaceous plant of 1- 6 feet high.The leaves are alternate petiole, 3–6 inches long, dullgreen, and rough, hairy, ovate or
8 rhombic with wavy margins. The flowers are small, with greenish or red terminal panicles. Taproot is long, fleshy red or pink. Theseeds are small and lenticellular in shape; with each seedaveraging 1 – 1.5 mm in diameter and 1000 seeds weighing 0.6 –
1.2g (Akubugwo et al., 2007). Amaranth are monoecious herbs (Mosyakin and
Robertson, 2003) which are primarily self-pollinated as female and male flowers are arranged in close proximity (Trucco and Tranel, 2011).
a.
9 b.
Plate 2.1
a. Basella albaleaves(http://luirig.altervista.org)
b. Amaranthus hybridus leaves(http://elqueliteuaaan.blogspot.com.ng/)
2.1.3 Botanical information.
Basella is a genus of the plant family Basellaceae. Basellaceae is a family of flowering plant in the order of Caryophyllales, with five genera consists of five species; one is pantropical and remaining four are widely distributed in East and Southeast Africa and
Madagascar (Sperling, 1987).At first Basella rubra and Basella albawere described to be two different species which were separated from each other on the basis of their stem colour and leaf character. As per the Echo Plant Information Sheet (2006), most authors agreed that the two colour forms of Basella are not separate species but one, these was supported by the facts that the anatomical differences in between these two taxa
10 are negligible (Deshmukh and Gaikwad, 2014). The cell, pollen morphological and protein profile studies of the red and green stem forms of Basellashowed that both the stem from Basella alba and Basella rubra belongs to one single species
(Mukherjee,2010).
TheAmaranthusgenus (Magnoliophyta: Caryophyllidae) comprises 70 species grouped into three subgenera (Mosyakin and Robertson 2003).Amaranthus hybridusis a basal species in the crop subgenus and conforms an interbreeding complex to two otherAmaranthus weeds:Amaranthus retroflexusand Amaranthus powellii(Trucco and
Tranel, 2011).
2.1.4 Classification
11
Basella alba L;
Kingdom Plantae – Plants
Subkingdom Tracheobionta - Vascular plants
Infrakingdom Streptophyta – land plants
Superdivision Tracheophyta – vascular plants, tracheophytes
Division Magnoliophyta
Class Magnoliopsida
Subclass Caryophyllidae
Order Caryophyllales
Family Basellaceae – Basella family
Genus Basella L - basella.
Species Basella alba L – Ceylon spinach (Kumar et al., 2013a;
Integrated Taxonomic Information System, 2015a).
12
Amaranthus hybridus L;
Kingdom Plantae- Plants
Subkingdom Tracheobionta – Vascular plants
Superdivision Spermatophyta – Seed plants
Division Magnoliophyta- Flowering plants
Class Magnolopsida – Dicotyledons
13
Subclass Caryophylidae
Order Caryophyllales
Family Amaranthaceae- Amaranth family
Genus Amaranthus L.
Specie hybridus– slim amaranth (Integrated Taxonomic Information
System, 2015b).
2.1.5 Chemical composition
Analysis of the dried leaves of Basella alba revealed the following composition (per 100g of the dried leaves); protein 20%, fat 3.5%, carbohydrate 54%, fibre 9% and ash 19%.
The leaves have been found to contain high level of calcium (3000mg) and are rich source of vitamin (Vit.) A (50mg), thiamine (B1)(0.7mg), Riboflavin (B2) (1.8mg), Niacin
(7.5mg) and vitamin C (1200mg), betacyanins, oxalic acid, flavonoids such as acacetin,
7,4-dimethoxy kaempferol and 4‘-methoxy isovitexin and some phenolic acids such as vanilla, syringic and ferulic acids (Daniel, 2006).
Amaranthus hybridus dried leaves composition (g/100g) was; 34.8% of protein, 9.6% of crude fat, 1.7% of crude fibre, 29% of carbohydrate and 1.7% ash (Adeyeye and
Omolayo, 2011). The leaves of Amaranthus hybridus have been found to contain high level of Mg (231 mg/100g) and Ca (44.15 mg/100g).The leaves are rich in vitamin C
14
(25mg/100g), vitamin A (β-carotene) (3.29mg/100g), Riboflavin (Vit. B2)(4.24mg/100g),
Thiamine (Vit. B1)(2.75mg/100g) (Akubugwo et al., 2007).
2.1.6 Uses
2.1.6.1 Culinary uses
In Nigeria, few rural and urban women cultivate few stands of Basella alba at their backyard for consumption and can be available throughout the year if well maintained.
The leaves and shoots of Basella are useful as vegetable which can be cooked as green or added to soups or with melon (Oloyede et al., 2013). The mucilaginous qualities of the plant make it an excellent thickening agent in soups, and stews.
The leaves, shoots and tender stems of Amaranthus hybridus are eaten as a potherb in sauces or soups, cooked with other vegetables, with a main dish or by itself.
The seed/ grain are also edible. Chopped Amaranthus hybridushave been used as forage for livestock. Amaranthus hybridus is not in the grass family, therefore it is not considered a cereal grain. However, it is used much like cereal grains, and is often called a pseudo cereal. Like other small grains, amaranth may be processed in popped, flaked, extruded and ground flour forms (Ouedraogo et al., 2011). The leaves and the softest portion of the shoots are usually boiled with water and then cooked with onions, tomatoes, oil and other additives of modern culinary delight. Amaranth leaves are
15 combined with other condiments to prepare soup. The leaves can also be dried and subsequently used during dry season (Masariramb et al., 2012).
2.1.6.2 Medicinal uses
Basella alba has been utilized for the treatment of various ailment.Leaves of B.alba is used for the treatment of hypertension in Nigeriaand treatment of malaria in Cameroon
(Anandarajagopal et al., 2011). It‘s also used in western Cameroon region to treat sexual asthenia and infertility in man (Nantia et al., 2011). The leaves of B. alba are traditionally used in ayurveda system of medicine to bring sound refreshing sleep when it is applied on head about half an hour before bathing (Kumar et al.,
2013a).Basella mucilage has been used in Thailand and Bangladesh traditional medicine in the treatment of bruise, ringworm, and acne. Leaves are used in constipation, poultice for sores, urticaria and gonorrhoea. It is also used in poultice local swellings, intestinal complaints (Yasmin et al., 2009).The leaf juice is used in Nepal to treat dysentery, catarrh and applied externally to treat boils. Basella alba has been used for the treatment of Anaemia in women, coughs, cold (leaf with stem), cold related infections mild laxative, diuretic and antipyretic (Chou, 1997;Akhteret al.,
2008;Rahmatullah et al., 2010). Root and leaves has been used for the removal of after birth, stomach pains and increase milk production (Pascaline et al., 2010). In India, it has been used for antipruritic and treatment of burn, it has also been used as anticancer such as melanoma, leukemia and oral cancer(Premalatha and Rajgopal, 2005; Saikia et al.,
2006).Daily consumption of Basella alba has been found to improvevitamin A stores in men (Focho et al., 2009). The plant has been reported for its antifungal, anticonvulsant
16 properties (Anandarajagopal et al., 2011).Maceration is taken orally for infertility, pelvic inflammatory disease, orchitis, epididymitis, threatened abortion, spurious labour (Focho et al., 2009).
Amaranthus hybridus is a highly nutritious plant, and contains all the essential amino acids (Akubugwo et al., 2007). It also provides an amazing amount of magnesium, calcium and dietary fiber for general health(Kadoshnikov et al., 2008).
Amaranthushybridus has been shown to contain large amount of squalene, a compound that has both health and industrial benefits (Akubugwo et al., 2007). Amaranthus hybridus is also used in the treatment of intestinal bleeding, diarrhoea and excessive menstruation(Omosun et al., 2008). The leaves of amaranth constitute an inexpensive and rich source of protein, carotenoids, vitamin C and dietary fibre (Shukla et al., 2006), minerals like calcium, iron, zinc, magnesium and phosphorus (Shukla et al.,
2006;Kadoshnikov et al., 2008). In Burkina Faso, Amaranthus hybridus is used in the treatment of liver infection, knee ache and the macerated aqueous extract is used as vermifuge. It is also used for it‘s also used as nematicides, diuretic and laxative in the children (Ouedraogo et al., 2011).
2.1.6.3 Industrial uses
The purplish sap from fruits of Basella alba is used as a colouring agent in pastries and sweets. (Ramu et al., 2011). The aqueous and methanol extracts of ripened fruit of
Basella albaare photo chemically unstable and thus can be used in the strong acid and strong base titrimetric reactions as an indicator (Mundo et al.,1995-96). Preliminary
17 investigations of Basella alba mucilage as a tablet binder in model drug paracetamol reported that, the mucilage exhibits good binding properties for uncoated tablets with small retardation in drug release from the tablet (Ramu et al.,2011).
Traditionally, sap from Amaranthus hybridusis used in dying and tanning of cloth (Jansen and Cardon, 2005).
2.2 Iron
2.2.1 Occurrence
Iron is an essential element required for the normal function of the body.It has an atomic number of 26 in the periodic table and has an atomic weight of 55.58. Iron is the fourth most abundant element and the second most abundant metal on earth. Ironis naturally available in many foods, added to some food products and available as dietary supplement. Iron servesas an essential component of haemoglobin, an erythrocyte protein that is essential for the transport of oxygen in the body from the lungs to the body tissues.
It also exist as component of myoglobin as a protein that provide oxygen to the muscles and it also exist as an integrated part of some enzyme systems in the body(Soetan et al.,
2010;Xin et al., 2015). Iron therefore is a very important micronutrient necessary for growth, development, normal cellular functioning, and synthesis of some hormones and connective tissues (Murray-Kolbe et al., 2010; Angett et al., 2012).
18
Iron exist in various oxidation state varying from Fe 6+ to Fe 2+ depending on its chemical environment. In aqueous environment of the body and in food iron exist in two oxidation states, ferrous (Fe2+) and ferric (Fe 3+) (Coates et al., 2010;Wessling-Resnick et al.,
2014).
2.2.2 Iron requirement and distribution in the body
The human body contains 2 to 4g of iron, or 38 mg/kg body weight for women and 50 mg/kg body weight for men. The difference in the iron content in both the male and the female may be attributed to the small iron reserve in women, lower concentration of haemoglobin and smaller vascular volume than men (Gropper and Smith, 2008). Over
65% of the total iron exist within the red blood cell as part of haemoglobin, 10% as part of myoglobin in the muscle and 1 to 5% are found as part of enzymes. The remaining body iron are stored in the liver, microphages of the reticulo-endothelial system and bone marrow (Munozet al., 2009).
Iron requirement vary with age and gender.It increases during growth period, pregnancy and for women having menstrual periods. Iron lost through cellular sloughing into the gastrointestinal tract, bleeding, and respiration cause loss of estimated 14µg/kg body weight/day. A non-menstruating 55 kg woman loses about 0.8 mg Fe/day and a 70-kg man loses about 1 mg Fe per day. The iron levels are restored by intestinal absorption of iron from the diet and internal iron turn over to maintain the balance (Bothwell,
2000;Arora and Kapoor, 2012).
19
2.2.3 Dietary iron
There are two forms of dietary iron; haem and non-haem iron. Haem iron refers to all forms of iron from animal source in which the iron molecule is tightly bound within the porphyrin ring structure, as it‘s found both in myoglobin and haemoglobin. Non-haem iron refers to all other forms of iron (Coates et al., 2010).In the humandiet, the primary sources of haem iron are the haemoglobin and myoglobin from consumption of meat, poultry, and fish whereas non-haem iron is obtained from cereals, pulses, legumes, fruits, and vegetables (Angett et al., 2012).Non-haem iron is the primary/main source of dietary iron. All dietary non-haem iron mixes together in the iron pool of the upper gastrointestinal tract.As a result of acidification in the stomach and subsequent exposure to pancreatic enzymes and gastrointestinal enzymes the inorganic iron is solubilized, ionized and reduced into ferrous form and kept soluble in the upper gastro-intestine by chelation to other compounds such as ascorbic acid and citrate. Diet also contains inhibitors of iron absorption such as phytate, polyphenol and tannins which are abundant in unprocessed cereals, indigestible fibre etc. These inhibitors binds to either the ferric or ferrous iron in the lumen of the gut forming insoluble precipitate making the iron unavailable for absorption. Haem iron is more highly bioavailable than non-haem iron, and its bioavailability is less affected by other components of the diet. Overall, dietary non-haem iron absorption is approximately 5–10% efficient compared with haem iron absorption at nearly 40%. Only about 10% of total dietary iron intake is represented by haem. However, given its greater absorption efficiency, haem iron plays a significant role in an individual‘s iron status (Coates et al., 2010).
20
2.2.4 Iron absorption
There are two major factors that determine the amount of iron that is absorbed into the body; the first is the total amount and form of iron compounds ingested and the second is the iron status of the individual (Hunt, 2005). Therefore individuals with higher iron status will absorb less amount of iron that is ingested through diet, and individuals with a lower iron status will absorb more of dietary iron. Almost all absorption of dietary iron occurs in the duodenum which involves several steps, including the reduction of iron to a ferrous state, tapered uptake, intracellular storage or transcellular trafficking and basolateral release. Both forms of dietary iron (haem and non-haem) are absorbed at the apical surface of duodenal enterocytes via different mechanisms(Pawelek and Masse,
2010).Both haem and non-haem iron have separate and distinct mode of uptake by the enterocyte.
2.2.4.1 Non-haem iron absorption
Dietary non-haem iron primarily exists in an oxidized ferric (Fe3+) form that is not bioavailable, and must first be reduced to the Fe2+(ferrous) form by a ferrireductase enzyme, duodenal cytochrome B, or dietary reducing agents such as ascorbic acid before it is transported across the intestinal epithelium by a transporter called divalent metal transporter 1 (DMT-1) (Muñoz et al., 2009). The ferrous iron then enters the enterocyte
21 via the DMT-1 where it can be stored as ferritin or be transported to the basolateral surface for entry into the circulation. Basolateral iron transfer occurs through the iron exporter ferroportin I (or metal transport protein-1), and the ferrous iron that is transported across the membrane is then oxidized by hephaestin before binding to plasma transferrin for transport throughout the body. Iron transfer across the basolateral membrane of the enterocyte is the rate-limiting step in iron absorption, endothelial cells receive a signal from the iron storage ―pools,‖ which then establishes a set point for iron absorption (Fleming and Sly, 2001). When there is excess iron in the cytosol, hepcidin, is released from hepatocytes into the plasma pool. Hepcidinbinds to ferroportin 1 causing its internalization and destruction (Ganz and Nemeth, 2006), thus inhibiting ferroportin 1 mediated export of iron. In cases of excess dietary or supplemental iron exposed to the microvillus enterocyte, the excess iron can be stored in the form of ferritin within the enterocyte (Coates et al., 2010).
The amount of ferritin that is synthesized by the enterocyte is under the regulation of the mRNA-binding protein, iron regulatory protein (IRP), which binds with high affinity at an iron response element (IRE) located in the 5‘-untranslated end of the ferritin mRNA.
There is also a similar set of IREs on the 3‘end of the mRNA for transferrin receptor
(TfR) and DMT-1 that allows for a reciprocal regulation of iron storage and iron uptake.
This IRE–IRP system of regulation, however, is also susceptible to oxidative stress, because nitric oxide may alter the affinity of this regulator of protein translation (Conrad and Umbreit, 2000). In situations of very high iron salt intakes, it is likely that an
22 increased oxidative stress within these enterocytes will lead to altered iron storage and absorption (Schuman, 2001).
2.2.4.2 Haem iron absorption
There are two prevailing hypotheses of haem iron absorption: receptor-mediated endocytosis and direct transport into the enterocyte throughhaem transporters. The hypothesis of receptor mediated endocytosis is a long-standing hypothesis that originated after discovery of a haem-binding protein in the upper small intestine in pigs and humans
(Grasbeck et al., 1979). Once the haem is internalized into the enterocyte, it is degraded by haem oxygenase 2 inside the vesicles that releases non-haem iron and produces bilirubin. The iron that is released from haem then needs to be transported out of the vesicle to join the labile iron pool. The transporter responsible for this has not yet been identified, but divalent metal transporter-1 (DMT-1) has been suggested as a possibility
(West and Oates, 2008). Once this iron joins the labile iron pool, it is processed in the same manner as dietary non-haem iron. The hypothesis that haem iron may be directly transported into the enterocyte originated after the recent discovery of haem transporters, proton-coupled folate transporter/haemcarrier protein 1 (PCFT/HCP1) (Shayeghi et al.,
2005 and Qiu et al., 2006) and feline leukemia virus subgroup C receptor-related protein
(FLVCR) (Quigleyet al., 2004).
Haem iron in the intestinal lumen is taken up by HCP1 directly into the cytoplasm and then has one of two possible fates. It can be catabolized to non-haem iron and bilirubin by
23 haem oxygenase 1 after which the non-haem iron joins the labile iron pool and is processed as non-haem dietary iron. Alternatively, intact haem may cross the enterocyte and be transported across the basolateral membrane by FLVCR(Coates et al., 2010).
2.2.5 Iron deficiency
Iron deficiency is one of the most prevalent nutrient deficiencies in the world, affecting an estimated two billion people worldwide (Stoltzfus et al., 2004).It is the most common type of anaemia that is characterized by the reduction or absence of iron (Fe) stores, low serum concentration of iron, poor haemoglobin concentration, haematocrit reduction and increased platelet count (Viteri 1993;Longo and Camaschella, 2015). It can result from inadequate iron intake, decreased iron absorption, increased iron demand and increase in iron loss (Short and Domagalski, 2013).
Iron deficiency anaemia has high prevalence in the developing countries,affecting over
30% of the world‘s population mostly infant, pregnant and postpartum women mostly from Africa and Asia. The high prevalence of iron deficiency anaemia in infant and pregnant women is due to high iron demandsof infant development and pregnancy.In resource-poor areas, iron deficiency anaemia is frequently exacerbated by infectious diseases (Stoltzfus et al., 2004; Adusi-Poku et al., 2008).
24
Iron deficiency has a considerable contribution to maternal morbidity by supressing the immune function with increased susceptibility to diseases or infections,reduced work capacity, performance and disturbance of postnatal cognition and emotions (Meiet al.,2005). Maternal iron depletion increases the risk of iron deficiency in the first 3 months of life, which can cause impaired mental development in infant and can negatively contribute to social emotional behaviours (Perez et al., 2005).
2.2.6 Major causes of iron deficiencyanaemia
2.2.6.1 Blood loss
Bloodloss is the most common cause of iron deficiency anaemia. Haemorrhage is the most common mechanism for acuteiron loss and anaemia.Itdecreases thehost‘s red cell mass, decreases the supply of ironfor erythropoiesis and increases the iron demand for erythropoiesis. Chronic blood lossfrom menstruation has the greatest impact globally(Miller, 2013).
Bleeding may occur from multiple sites along the intestinal tract, with an increased incidence of bleeding from the colon(Lanas et al., 2009). Less than 2 ml of blood is lost daily in the stool of healthy adults (Ahlquist et al., 1985). Detection of minute blood losses of up to 60 ml/dmay be difficult without specialized stool tests(Rockey, 1999).
Other causes of blood loss include blood donation and nosebleeds. Afterchronic physical
25 exertion, significant iron islost in sweat and may contribute to the deficientstate (Reinke et al., 2010).
2.2.6.2 Maternal– fetal bridge of iron deficiency
Iron requirements increases around thetime of birth and during menstruation in females.
During pregnancy, it is estimated that approximately 1200 mg of iron arerequired from conception through delivery (Leeand Okam, 2011). Iron intake and stores in themother must satisfy fetal development, andblood loss at delivery. Additionally, the maternal erythrocyte mass should increase from 350to 450 ml. By comparison, pregnant womenwithout iron supplements only increase theirred cell mass by 180– 250 ml
(Pedersen andMilman, 2003). During postpartum, iron is lost as lactoferrin in breast milk,those losses are balanced by the absence ofmenstruation in the lactating female.
Maternal iron deficiency anaemia duringpregnancy and the perinatal period have devastating effects on both the mother and child. Inaddition to the direct effects of anaemia, reduced fetal brain maturation, paediatric cognitivedefects, and maternal depression are associatedwith iron deficiency anaemia (Black et al., 2011).
The reversibility of cognitive defects caused at an early age by iron deficiency is unclear.
Importantly, untreated iron deficiency in pregnantfemales will be passed to the infant. If left untreated during infancy, childhood, and adolescence, anaemia and iron-associated cognitivedefects may conceivably be passed betweengenerations much like genetic traits.
26
Unlessthe iron deficiency is treated at some stage oflife, the cycle of iron deficiency from motherto child may remain unbroken for several generations (Miller, 2013).
In the fetus and during infancy, iron is required for the growth and development of all tissues. The human growth rate is almost logarithmic during this period (Anderson and
Holford, 2008). In anaemic mothers with iron deficiency,the fetal ferritin levels remain
10 times higherthan the maternal ferritin at the time of delivery(Erdem et al., 2002). At birth, the fetal red cellmass is approximately 50 ml/kg (Phillips et al., 1986), compared with 25– 30 ml/kg in adults (Fairbankset al., 1996). During the first year of postnatal life,total-body iron increases by approximately 240 mg (Oski, 1993). Around 80% of that iron is used for expanded haemoglobin production (50%) and ironstores (30%). Beyond the first year, iron intake orstores must remain sufficient to support the ongoing growth and increased red cell mass (Moseret al., 2011). In children and young adults, irondeficiency remains one of the top three contributors to the overall disease burden in those populations (Gore et al., 2011).
2.2.6.3 Malaria
Iron deficiency anaemia and malaria coexist in most tropical regions of the world.
Malaria contributes to iron deficiency anaemia by causingintravascular haemolysis with subsequent lossof haemoglobin iron in the urine. Malaria also causes an immune response that suppresses erythropoietin(Burgmann et al., 1996), as well as direct effectson erythropoiesis (Skorokhod et al., 2010). Thehost may also increase hepcidin expression
27 forprotection from liver-stage malaria (Portugalet al., 2011). Increased hepcidin restricts iron and might delay erythroid recovery.(Sazawal et al., 2006).
2.2.6.4 Hookworm
Like iron deficiency anaemia, hookworm infection affects several hundred million humans globally (Bungiro and Cappello, 2011). A recent study reported that there is aconsiderable overlap between malaria and hookworm in sub-Saharan Africa (Brookeret al., 2006). Worldwide, there are two hookwormspecies that infect humansAncylostoma duodenale and Necator americanus, are both found intropical regions due to the favorable condition that is necessary for their growth and survival. The worm is introducedto the soil by fecal matter in regions where sanitation is not present.
Ahookworm infection should be suspected incases of travel or habitation in the tropics, irondeficiency anaemia, and mild eosinophilia. Owing to their location in the small bowel, capsuleendoscopy is helpful for diagnosis if eggs are notpresent in the stool (Li et al., 2007).
Iron-deficiency anaemia resulting from hookworm infection is caused due to chronic intestinal blood loss and often causes long-term morbidity (Albonjco et al., 1998;
Steketee, 2003). Blood loss is caused primarily by parasite release of coagulases, causing ongoing blood loss in the stool, rather than actual blood consumption by the parasite. For example, Ancylostoma duodenale is estimated to cause up to 0.25 mL of blood loss per worm per day (Steketee, 2003). Hookwormslive on 0.3– 0.5 mL of blood extravasated dailyfrom the intestinal mucosa. Heavily infected patients are simply unable to maintain
28 adequateiron stores and become anaemic (Smith andBrooker, 2010). Even without additional ironsupplements, anti-helminthic drugs can causereversal of iron deficiency anaemia (Radhika et al.,2011).
2.2.7 Some haematological parameters and their role in diagnosis of iron deficiency anaemia
2.2.7.1 Haemoglobin concentration
One recognised method for the diagnosis of iron deficiency in individuals or populations involves monitoring changes in haemoglobin (Hb) concentration. It‘s measured as part of complete blood count from a blood sample (WHO, 2001). The world health organisation defines the cut-off for iron deficiency anaemia as blood haemoglobin value of <7.7 mmol/L (<13 g/dL) and <7.4 mmol/L (<12 g/dL) in male (Bermejo and Garcia-Lopez,
2009).
Haemoglobin is an iron-containing oxygen-transport metalloproteins that transport oxygen to body tissues and transport carbon dioxide from the tissues to the lungs.
Haemoglobin is a spheroidal haem protein having four subunits, each consisting of a globular protein non-covalently bound with an embedded haem group. Haemoglobin has a molecular weight of about 64456 kDa.The globular protein units of Hb is made up of two identical pairs of polypeptide chains, i.e. two identical alpha (α) chains containing
141 amino acids and two identical non- α chains (beta (β), gamma (γ),delta (δ) or epsilon
29
(ε) chains). In adult humans the non- α chains are beta (β), containing 146 amino acids
(Nelson and Cox, 2005).
The haem group consists of iron (Fe2+) ion held in a heterocyclic ring, known as aporphyrin. This porphyrin ring consists of four pyrole molecules cyclically linked together (by methene bridges) with the iron ion bound in the centre (Casiday, 2000).
When oxygen is not bound, a very weakly bonded water molecule fills the site, forming a distorted octahedron. Even though carbon dioxide is also carried by haemoglobin, it does not compete with oxygen for the iron-binding positions, but is actually bound to the protein chains of the structure (Linberg et al., 2009).
2.2.7.2 Serum ferritin concentration
Ferritin is considered the most important indicator of iron status and a central parameter for determination of therapeutic response (Mei et al., 2005; O‘Meara et al., 2011). Serum ferritin is the first parameter to reflect iron deficiency even when the haemoglobin concentration, haematocrit level and serum iron are normal (Knovich, et al., 2009; Short and Domagalski, 2013) . Therefore serum ferritin is an important biochemical parameter for monitoring iron deficiency in the absence of inflammation and infection and also it can be sued to differentiate between anaemia of chronic disease with iron deficiency
(Walker et al., 2010).
30
Ferritin is the major protein involved in the storage of iron.It is approximately 450 kDa protein comprising 24 apoferritin monomers that associate to form a hollow spherical particle. Up to 4000 atoms of iron can bind in the sphere where they are stored as Fe3+ ions. In human cells there are two subunits of ferritin exist; light (L) and heavy (H).Most tissue ferritin molecules are a heterogeneous mixture varying proportions of the two subunits. Circulating ferritin is normally predominantly in the L form and is not iron‐bearing. The H subunit is involved in iron transport, while the L subunit is responsible for long-term storage of iron in the liver and spleen (Vanarsa et al., 2012).
Apart from being a storage protein, ferritin also plays an important role in body immune response as it‘s evident from increased concentration during infection, and inflammation.
Therefore the two key factors that regulate ferritin expression are iron and inflammatory cytokines (Muntaneet al., 1995; Walker et al., 2010).
Ferritin concentration vary by age and sex. It is high during the period of birth, rise during first two month of life and decrease during the latter part of infancy(Domellöf et al., 2002). At about age one, concentrations begins to rise again and continue to rise into adulthood. During adolescence, however, males have higher ferritin concentration than females, a trend that persists into late adulthood. Values among men peak between 30–39 years of age and then tend to remain constant until about 70 years of age.Among women, serum ferritin values remain relatively low until menopause and then rise (Gibson, 2005).
31
Ferritin is usually assessed in serum or plasmawith enzyme-linked immunosorbent assays
(ELISA) orenzyme immunoassays after venous blood collection. However dried serum spot samples can also be used (WHO, 1989).
2.2.7.3 Total iron binding capacity and transferrin saturation.
Total iron binding capacity (TIBC) measures all of the proteins in the blood that are available to bind with iron, including transferrin.Total iron-binding capacity (TIBC) indicates the maximum amount of iron needed to saturate plasma or serum transferrin
(TRF), which is the primary iron-transport protein. The body produces transferrin in relation to the need for iron. When iron stores are low, transferrin levels increase and vice versa(Yamanishi, 2003).
2.2.7.4 Serum transferrin receptor (stfr)
Soluble transferrin receptor (sTfR) protein is a single polypeptide chain of 85 kDa that can be measured in human serum, derived from transferrin receptor, a transmembrane cellular protein of 190 kDa, primarily expressed in cells that require iron. Transferrin receptor is composed of 2 disulfide-linked monomers of 95 kDa, each containing 760 amino acids, and organized into 3 major portions: a large C-terminal extracellular domain of 671 amino acids, a transmembrane domain of 28 amino acids and an N-terminal cytoplasmic domain of 61 amino acids. A proteolytic cleavage by a matrix metalloproteinase between arginine-100 and leucine-101 of the extracellular domain
32 forms the sTfR (Speeckaert et al., 2010). Plasma sTfR concentration reflects the receptor density on cells and the number of cells expressing receptors.Therefore, it is closely related to cellular iron demands and to erythroid proliferation rate (Skinke, 2008).
Serum transferrin receptor concentration has been shown to be unaffected by chronic disease and inflammation.Therefore it is an important biochemical parameter in identifying iron deficiency anaemia (Ong et al., 2008; Berlin et al., 2011; WHO, 2011).
2.2.7.5 Serum iron concentration
Serum iron measures the amount of iron that is bounded to the transferrin in the serum. A normal serum iron is 55 – 155 ug/dL. Lower levels may indicate iron-deficiency anaemia or anaemia of chronic disease, while higher levels may indicate haemolytic anaemia or vitamin B12 deficiency.
2.2.8 Prevention of iron deficiency
Prevention of both iron deficiency and anaemia require approaches that address all the potential causative factors. Interventions to prevent and correct iron deficiency and iron deficiency anaemia, therefore, must include measures to increase iron intake through food-based approaches, namely dietary diversification and food fortification with iron; iron supplementation and improved health services and sanitation.
33
2.2.8.1 Dietary diversification
Dietary diversification is encouraging the increase in consumption of micronutrient rich foods in both quantity and the range of foods such as dark green leafy vegetables, lentils and vitamin C rich fruits which may be available but are underutilised by the deficient population. Increasing dietary diversity has a potential of improving the general nutritional status of the population because it improves the consumption of other food constituent not just micronutrient (Allen et al., 2006).
2.2.8.2 Food fortification
Food fortification has a long history of use in the industrialised countries in the control of vitamins deficiencies such as vitamin A, D and several B vitamins and micronutrients such as iodine and iron (WHO/FAO, 2006). Food fortification is a useful public health intervention if the food to be fortified iswidely consumed in sufficient amounts by at-risk individuals and if the fortificants do not have a toxic or unappealing effect on the food or significantly affect food price. In many situations, this strategy can lead to relatively rapid improvements in the micronutrient status of a population, and at a very reasonable cost, especially if advantage can be taken of existing technology and local distribution networks(Allen et al., 2006).
Ferrous sulfate and fumarate are commonly used in food fortification, they have similar and high bioavailable iron content. Ferrous sulfate is cheapest and widely used to fortify
34 flour but may cause rancidity with storage. Insoluble compounds (ferric pyrophosphate and orthophosphate) have less reactivity with food but poorer bioavailability. Elemental iron compounds have been used but have poor bioavailability and affect taste at effective concentrations. Fortification of cereals, salt, and sauces has been successfully achieved using iron EDTA, which contains iron protected by a chelating moiety that can also improve absorption of intrinsic non-haem dietary iron(Pasricha et al., 2013).
2.2.8.3 Supplementation
Food supplements are highly concentrated vitamins and minerals producedin the form of capsules, tablets or injections and administered as part of health care or specific nutrition campaigns (UNICEF, 1998). Food supplementation has the advantage of being capable of supplying an optimal amount of a specific nutrient or nutrients, in a highly absorbable form, and is often the fastest way to control deficiency in individuals, population or groups that have been identified as being deficient. In developing countries, supplementation programmes have been widely used to provide iron and folic acid to pregnant women, and vitamin A to infants, children under 5 years of age and postpartum women (Allen et al., 2006).
2.3 Blanching
Blanching is a technique that is performed prior to freezing, canning, or drying in which fruits or vegetables are heated for the purpose of inactivating enzymes; modifying
35 texture; preserving colour, flavour, and nutritional value; and removing trapped air. (De
Corcuera et al., 2004).
Water at high temperature or steam are the most commonly used for blanching which can be responsible for quality deterioration of processed vegetables (Shivhare et al., 2009;
Sanjuan et al., 2005). Also, similarly to other thermal processes, blanching have been shown to affect the content of some nutritive and bioactive components of vegetable such as vitamin C (Sablani, 2006). However, blanching can also modify food attribute such as flavour which can be due to a substantial decrease of concentration of important volatile compounds after blanching (Soria et al.,2008).
2.4 Antinutrients
2.4.1 Antinutrtional factors
Antinutrtional factors or antinutrients are natural or synthetic substances that interfere with nutrient intake, digestion, absorption and utilization which hamper with the normal body function as it cannot absorb nutrient at optimum level.Plants generally acquired antinutrients from fertilizer or from several natural occurring chemicals. Some of these chemicals are known as ‗‗secondary metabolites‘‘ and they have been shown to be highly biologically active (Zenk, 1991). Several antinutrients like proteinase inhibitors, lectins, phytates, polyphenols, other are more specific, as some complex glycosides. Most of food antinutrients have an impact on the digestive system, like the inhibition of digestive
36 enzymes (e.g.protease inhibitors), impairment of hydrolytic functions and of transport at the enterocyte site (lectins), formation of insoluble complexes which cannot be adsorbed, decrease of bioavailability of some nutrients (phytates, polyphenols), and the increase of the production of gases in the colon (α-galactosides) (Thompson, 1993).
2.4.2 Biochemical effects of someanti-nutritional factors
2.4.2.1 Phytate
Phytate, myo-inositol hexakisphosphate, is the salt of phytic acid and is widely
distributed in all seeds and possibly all cells of plants and can reach concentration higher
than 10% of dry matter(Bora, 2014). It serves as a storage of phosphorous and minerals
and accounts for 60-90% of the phosphorous in the plant (Harland and Morris, 1995).
Phytates bind minerals like calcium, iron, magnesium and zinc and make them
unavailable (Soetan and Oyewole, 2009). Anaemia and other mineral deficiency
disorders are common in regions where the diet is primarily a vegetarian (Erdman, 1995).
2.4.2.2 Oxalate
Oxalate is a salt of oxalic acid which is a strong organic acid which is widely distributed
in nature in both plant and animals but more predominant in plant (Liebman, 2002).
Oxalate affect the absorption and metabolism of calcium and magnesium. It also react
with protein to form complexes which have inhibitory effect in peptic digestionand can
increase the risk of developing kidney stones (Holmes et al., 2001).
2.4.2.3 Saponins
Saponins are a heterogenous group of naturally occurring glycosides of high molecular
weight. Saponins occur widely in plant species and exhibit a range of biological
properties. Saponins are group of natural products possessing the property of
37 producing lather or foam when shaken with water and their ability to haemolyse red blood cell (Jenkins et al., 1994). Saponin are diverse class of glycosides found mainly, but not exclusively in plants, they comprises a steroidal or triterpene aglycone linked to one, two or three saccharide chains of varying size and complexity via ester and or ether linkages. Saponins occurs in broad range of plants consumed in the human diet, including legumes (soy, peas and beans), root crops (potato, yam, asparagus and allium) as well as in oats, sugarbeet, tea and many medicinal herbs such as ginseng (Price et al., 1987). Saponins reduce the uptake of certain nutrients including glucose and cholesterol at the gut through intralumenal physicochemical interaction. Hence, it has been reported to have hypocholesterolemic effects (Umaru et al., 2007).
2.4.2.4 Tannins
Tannins are water soluble polyphenols that are present in many plant food. Tannins bind to proteins through hydrogen binding and hydrophobic interactions, thereby reducing their nutritional quality and combine with digestive enzymes thereby making them unavailable for digestion (Abara et al., 2003). They also cause decreased palatability and reduced growth rate (Roeder, 1995).
2.4.2.5 Cyanogenic glycosides
Cynogens are glycosides of 2-hydroxyl nitriles and widely distributed among plants.
When plants are wounded by herbivores or other organisms, the cellular compartmentation breaks down and cynogenic glycosides come into contact with active β-glucosidase, which hydrolyses them to yield 2-hydroxynitrile. In addition to
38 the toxic effects, cynogens it can serve as mobile nitrogen storage compounds in seeds which are important during germination. Some legumes like kidney bean, red gram and linseed contain cyanogenic glycosides from which hydrogencyanide (HCN) may be released by hydrolysis (Akande et al., 2010) HCN can cause disfunction of the central nervous system, respiratory failure and cardiac arrest ( D‘Melloet al., 1991)
2.4.2.6 Lectins
Lectins are also called phytohaemagglutinins. Lectins are glycoproteins widely distributed in legumes and some oil seeds (including soy bean) which possess affinity for specific sugar molecules and are characterized by their ability to combine with carbohydrate membrane receptors (Pusztai, 1989). Lectins have the capability to directly bind to the intestinal mucosa interacting with the enterocytes and interfering with absorption and transportation of nutrients particularly carbohydrates during digestion
(Santiago et al., 1993) and causing epithelial lessions within the intestine ( Oliveira et al., 1989).
2.4.2.7 Protease inhibitors
Protease inhibitors are widely distributed within the plant kingdom. Protease inhibitors have the ability to inhibit the activity of proteolytic enzymes within the gastrointestinal tract of animals (Liener and Kakade, 1980). Trypsin inhibitors irreversibly bind trypsin, making the enzyme incapable of performing its role in the breakdown of proteins. This causes the intestine to release cholecystokinin to stimulate the pancreas to enlarge. The amino acids present in trypsin cannot be reabsorbed and thus are lost when the trypsin combining with the trypsin inhibitors (Karl et al., 1987).
39
40
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Materials
3.1.1 Chemicals
All chemicals and reagents used were of analytical grade.Ferritin rat (Enzyme-Linked
Immunosorbent Assay) ELISA kit was purchased from United Kingdom. Other chemicals were purchased from Steve-Moore chemicals store in Zaria, Kaduna State,
Nigeria.
3.1.2 Collection of plant materials
Basella alba and Amaranthus hybridus vegetables were harvested from their natural habitats during rainy season.Basella alba was harvested from Ikoro-Ekiti, Ekiti state, while Amaranthus hybridus was harvested from Samaru-Zaria, Kaduna state. The two
41 vegetables wereidentified and authenticated in the Department of Biological Sciences,
Ahmadu Bello University, Zaria where vouchers number;2225 and 20785were depositedrespectively.
3.1.3 Animals
Forty eight (48) 3 week‘s old Albino ratsof both sexes were obtained and kept in well aerated plastic cages in the animal house, Department of Pharmacology and Therapeutics,
Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria. The animals were acclimatized in the animal house environment for a period of two weeks before the commencement of the study. They were fed with grower‘s mash and water ad libitum.
3.2 Methods
3.2.1 Preparation of the vegetables
The harvested vegetables were washed in deionized water. They were divided into two part, the first part was air dried at room temperature, grinded and storedfor further use while the second part was blanched.
42
3.2.2 Blanching of the vegetables
Both vegetable were blanchedseparately in deionized water at 100oC for 5 minutes. The blanched vegetables were dried under room temperature and grinded. They were stored for further use.
3.2.3 Quantitative determination of vitamin C
Vitamin C content was determined according to the method described by Badrakhanet al.,
(2004). Plant sample (0.05g) was weighed and transferred into a test tube. Methanol
(25ml) was added and the mixture was allowed to stand for 10min to extract properly.
The sample was filtered, 1ml of the filtrate was transferred into a cuvette and 3ml of the extracting solvent (Methanol) was added. Vitamin C concentration was determined using the UV spectrophotometer at 478.5nm wavelength (Shimadzu 2550 Model).
3.2.4 Quantitative determination of some antinutrients
3.2.4.1 Phytic acid determination
This was determined using the procedure described by Lucas and Maskakas (1975). Each sample (2g) was weighed into 250ml conical flask, 100ml of 2% concentrated HCl was used to soak each sample for 3hrs. The mixture was filtered and 50ml of each filtrate was placed in 250ml beaker and 107ml of distilled water was added to each solution, 10ml of
0.003M thiocyanate solution was added as indicator and then titrated with standard iron chloride solution which contains 0.00195 iron per ml.
% Phytic acid= Y × 1.19 × 100
43
Where Y = Titre value × 0.00195
3.2.4.2 Oxalic acid determination
This was determined using the titration method described by Day and Underwood (1986).
Sample (1g)was weighed into 100ml conical flask where 75ml of 3M H2SO4was added and stirred for 1hr. It was then be filtered using whatman No 1 filter paper. From the
0 filtrate, 25ml was taken and titrated while hot (80-90 C) against 0.1M KMnO4 solution until a faint pink colour persist for at least 30 seconds. The overall redox reaction is:
2 + 2+ MnO4 + C2O4 + 8H Mn + 4H2O + 2CO2
Oxalate (mg/100g) = T × Vme × D.F × 100/Me × Ms (g)
Where
T = Titre value of KMnO4 (ml)
3 Vme= Volume mass equivalent (1cm of 0.05M KMnO4 solution is equivalent to 0.00225
anhydrous oxalic acid)
D.F = Dilution factor (Vt/A =75/25= 3)
Where
Vt = Total volume of titrate( filtrate 75ml) and
A = Aliquot used (25ml)
44
Me = Molar equivalent of KMnO4 redox reaction
Ms = Mass of sample used
3.2.5 Quantitative determination of mineral content
Mineral content was determined by method of AOAC, (2000)
Principle
The method involves the separation of minerals from the food matrix by destruction of the organic matter of the sample through wet digestion. The mineral content (Ca, Cu, Fe,
K, Mg, Na, Zn) in diluted acid is then determined by atomic absorption spectrophotometer, (AAS)
3.2.5.1 Sample digestion (Wet)
Powdered plant sample (0.2g) was weighed into the digestion tube and 5ml of HNO3-
HCLO4 (2:1 by volume) mixture was added and mixed. The tube was placed into the digestion block inside a fume hood, the temperature was set at 1500C and digestion was done for 1hr 30min. The temperature was later increased to 2300C and digested for another 30 min (white fuming stage). The temperature was reduced to 1500C, 1ml of
HCL was added and heated for 30min. The digester was switched off and allowed to cool to room temperature, water was added to get to 50ml mark on the tube. The concentration of Fe, Mg, Zn, Cu and Ca was read using the flame atomic absorption spectrophotometer
(FAAS), AA 500 model.
45
3.2.6 Determination of the speciation pattern of iron
The speciation pattern of iron in the vegetables was determined using the method of
Goran et al., (1999).
3.2.6.1 Exchangeable phase
Magnesium chloride (20ml) of 1M solution pH 7, was added to 1g of the dry sample, the mixture was stirred continuously at 1 hr room temperature, centrifuged at 1500 xg for
30min, filtered and the liquid extract was analysed for iron using the FAAS.
46
3.2.6.2 Carbonate phase (exchangeable phase)
Sodium acetate (20ml) of 1M solution pH 5 (adjusted with acetic acid) was added to the residue from step 1, the mixture was stirred continuously at 6hr room temperature and then centrifuged at 1500 xg for 30min, filtered and the liquid extract was analysed for iron using FAAS.
3.2.6.3 Amorphous Fe oxyhydroxide phase (reducible)
Hydroxylamine Hydrochloride (20ml) of 0.25M solution was added to the residue from step 2, the mixture was heated at 600C for 2hr vortexed and centrifuged at 1500 xg for
30min, filtered and the liquid extract was analysed for iron using the FAAS.
3.2.6.4 Crystalline Fe oxide phase (reducible)
NH2.HCl of 1M(30ml) was added to the residue from step 3 and was heated for 3hr at
900C and vortexed 9 times, then centrifuged at 1500 xg for 30 min, filtered and the liquid extract was analysed for iron using FAAS
3.2.6.5 Sulphides and organic phase (oxidizable)
Potassium trioxochlorate (V) (750mg) and Hydrochloric acid (5ml) of 12M solutionswere added to the residue from step 4 and vortexed. Hydrochloric acid (10ml) of 12Msolution was added to the mixture then vortexed and left for 30min. 15ml of deionised water was added and vortexed again. Finally, trioxonitrate (V) acid (10ml) of 4M solution was
47 added and vortexed. The mixture was heated for 20min at 900C, centrifuged at 1500 xg for 30min and the liquid extract was analysed for iron using FAAS.
3.2.6.6 Silicates and residual phase
Trioxonitrate (V) acid solution (2ml) of 16M was added to the residue from step 5 and heated to dryness at 2000C then cooled. Hydrochloric acid (2ml) of 12M solution was added and heated. Aqua regia solution (10ml)was added and the mixture heated to dryness at 1200C then hydrochloric acid (1ml), trioxonitrate(V) acid (3ml) and 3ml of deionised water were added and warmed. The mixture was centrifuged at 1500 xg for
30min, then the liquid extract was analysed for iron using FAAS
3.2.6.7 Invitro bioavailable iron
This was obtained by summing the exchangeable, oxidizable and reducible iron content of the vegetables.
Therefore percentage bioavailability = x 100
Where; EXC = Exchangeable fraction
OXI = Oxidizable fraction
RED = Reducible fraction
RES = Residual fraction
48
3.2.7Diet formulation.
The Iron deficient diet formulation was done according to the method of Yi Ning et al.,(2002).
Table 3.1 Diet Composition of Iron Deficient and Iron Sufficient Diet
Ingredient Iron sufficient diet (g/kg) Iron deficient diet (g/kg)
Corn starch 625 625
Casein 200 200
Fibre 50 50
Soybean oil 70 70
Mineral mix 35 35
Vitamin mix 10 10
Ferrous sulphate 10 0
3.2.8 Induction of anaemia
49
Iron deficiency anaemia was induced by feeding normal rats exclusively on the iron deficient formulated diet for a period 14 weeks. Haemoglobin (Hb) concentration was monitored at two weekly interval and used as index for anaemia. Rats with Hb concentration of <11g/dl were considered to be iron deficient (Velmurugan et al.,2010).
3.2.9 Animal grouping
The experimental rats were divided into 8 groups, with 6 animals per group.
• Group 1: Normal rats (non-anaemic) fed on iron sufficient diet and deionized
water ad libitum.
• Group 2: Anaemic rats fed on the iron deficient diet and deionized water ad
libitum.
• Group 3: Anaemic rats fed on iron sufficient diet and deionized water ad libitum.
• Group 4: Anaemic rats fed on iron deficient diet supplemented with 10%
unblanchedBasella alba and deionized water ad libitum.
50
• Group 5: Anaemic rats fed on iron deficient diet supplemented with 10%
blanched Basella alba and deionized water ad libitum.
• Group 6: Anaemic rats fed on iron deficient diet supplemented
with10%unblanched Amaranthus hybridus and deionized water ad libitum.
• Group 7: Anaemic rats fed on iron deficient diet supplemented with 10%
blanched Amaranthus hybridus anddeionized water ad libitum.
• Group 8: Anaemic rats given iron deficient diet with ferrous sulphate drug
(2.78mg/kg body weight) administered orally anddeionized water ad libitum.
Diet Supplementation was carried out for 21 days.
3.2.10 Collection of blood sample
During induction and supplementation 1ml of blood was collected into EDTA
(Ethylenediaminetetraacetic) bottle through the tail vein. At the end of supplementation the rats were anesthetized using chloroform and immediately sacrificed. Blood was collected into EDTA and dry test tubes. The blood in the dry tubes were allowed to clot.
They were then centrifuged at 1000 xg for 10 minutes then supernatant (serum) was collected and stored in a freezer for further analysis.
3.2.11 Determination of haematological parameters
51
3.2.11.1 Packed cell volume
Thiswas determined weekly by the microhaematocrit method as described by Dacie and
Lewis (2001)
Principle: The red blood cells are heavier than plasma with specific gravity of 1090 and
1030 respectively. When blood is placed in a capillary tube and centrifuged, they settle and pack because of the centrifugal force acting on them. The volume occupied by the cells is measured and its ratio with that of the volume of the whole blood is calculated.
Procedure: Blood from EDTA bottle was allowed by capillary action to flow through the capillary tube and one end of the tube was sealed by flaming. It was then centrifuged at10,000 x g for 5 minutes. The PCV was estimated using a microhematocrit reader and expressed as percentage erythrocytes the blood contain.
3.2.11.2 Haemoglobin concentration
This was determined by the cyanomethaemoglobin method (Jain, 1986)
Principle:Blood is mixed with dilute hydrochloric acid. This process haemolyses the red cells, disrupting the integrity of the red cells membrane and causing the release of haemoglobin, which, in turn, is converted to a brownish-colored solution of acid haematin. The acid haematin solution is then compared with a colour standard.
Procedure: Sample solutions and standard solutions were prepared as follows.
Random sample: 5000 µl of Drabkin reagent was mixed with 20 µl of distilled water
52
Standard: 5000 µl of Drabkin reagent was mixed with 20 µl of sample solution of haemoglobin
Target sample: 5000 µl of Drabkin reagent + 20 µl of blood
The concentration of haemoglobin was marked with Drabkin‘s method, with the use of a spectrophotometer, at 540nm wavelength. Once Drabkin reagent is mixed with the blood, the solution was incubated at room temperature for the duration of 5 minute and absorbance was measured. The spectrophotometer was set to zero using distilled water.
Measurement formula: Haemoglobin and its derivatives affected by Drabkin reagent transform into Cyanomethaemoglobin, whose optical density was measured photometrically.
The concentration of haemoglobin was calculated according to the following formula:
3.2.11.3 Red blood cell (RBC) and white blood cell (WBC) count
Red blood cell (RBC) and white blood cell (WBC) count were determined using the improved Neubauer counting chamber (Dacie and Lewis, 2001). Improved Neubeur haemocytometer (counting chamber) was used to count both white blood cells (WBCs) and red blood cells (RBCs) under the light microscope.
53
Principle: Blood is diluted with a fluid containing glacial acetic acid that lyses the red cells but not the white cells and a dye (gentian violet) which stains the nucleus of the white cells to facilitate counting.
Procedure: The white blood cell and red blood cell pipettes was used to draw blood and fill to 0.5 mark of both WBC and RBC pipettes. WBC diluting fluid was drawn to 11 and
101 marks respectively. The fluid and the blood were then mixed gently and transferred into the counting chamber. It was allowed to settle for 2mins and the chamber placed on the stage of the microscope for counting. The white blood count was done using the x10 objective while the red blood cell counts was done using the x 40 objective.
Calculation: Number of cells counted divided by volume of one square multiplying number of squares counted and multiply by dilution factor.
3.2.11.4 Determination of mean corpuscular volume, mean corpuscular haemoglobin,
and mean corpuscular haemoglobin concentration.
These were calculated by the method of Dacie and Lewis, (2001).
Mean corpuscular Volume (MCV) in fL was calculated as:
Mean corpuscular haemoglobin (MCH) in pg was calculated as:
54
Mean corpuscular haemoglobin concentration (MCHC) in g/dl was calculated as:
3.2.12Determination of serum ferritin concentration
Serum ferritin level, an in-vivoindex for iron was determined using the Abcam‘s Ferritin
(FTL) Rat ELISA kit according to the manufacturer‘s protocol.
Principle: The ferritin present in samples reacts with the anti- ferritin antibodies which have been adsorbed to the surface of polystyrene microtiter wells. After removal of unbound proteins by washing, anti-ferritin antibodies conjugated with horseradish peroxidase(HRP), are added. These enzyme labelled antibodies form complexes with the previously bound ferritin. Following another washing step, the amount of enzyme bound in complex is measured by the addition of a chromogenic substrate, 3,3‘5,5‘- tetramethylbenzidine (TMB). The quantity of bound enzyme varies proportionately with the concentration of ferritin in the sample tested; thus, the absorbance, at 450nm, is a measure of the concentration of ferritin in the test sample. The quantity of ferritin in the test sample can be interpolated from the standard curve constructed from the standards, and corrected for sample dilution.
55
Assay procedure:100µl of each standard including zero control, and 100µl was pipetted into pre designated wells, the microtiter plate was then incubated at room temperature for
60 minutes (the plate was kept covered and level during the incubation) and then the contents of the well was aspirated using diluted wash buffer. 100µl of appropriately 1x
Enzyme-Antibody Conjugate was pippeted into each well and incubated at room temperature for ten (10±2) minutes, the plate was kept covered and level in the dark during incubation, the wells were then washed by aspirating the contents of the wells using diluted wash buffer. 100µl of TMB Substrate solution was then pipette into each well, this was incubated in the dark for precisely ten(10) minutes afterwhich 100 µl of stop solution was added to each well. The absorbance of each well content was read at
450nm using the microplate reader.
Calculation: Control blank reading was subtracted from all the standard readings. The standard readings were plotted against their concentrations and the best smooth curve through these points was drawn to construct the standard curve. Protein concentrations for the samples were then extrapolated from the standard curve plotted (Appendix 9).
3.3 Statistical Analysis
Data was analysed using statistical package for the social sciences (SPSS), version 20.
The results were expressedas mean ± standard deviation (SD) except where otherwise stated. The data was analysed by analysis of variance (ANOVA). The difference between the various animal groups were compared using the Duncan Multiple Range Test. P values less than 0.05 (p<0.05) were taken as significant.
56
CHAPTER FOUR
4.0 RESULT
4.1 Vitamin C Content of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves
Result of the vitamin C content of unblanched and blanched Basella alba and
Amaranthus hybridus leaves is presented in Figure.4.1. The result shows that Amaranthus hybridus leaves has significantly (p<0.05) higher vitamin C concentration than Basella alba leaves and there was a significant (p<0.05) reduction in the vitamin C content of
57
both the leaves after blanching, with the blanched Basella albaleaves having the lowest
vitamin C content.
4.2 Antinutrients Content of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves
Antinutrients content ofblanched and unblanched Basella alba and Amaranthus hybridus leaves are presented in Figures 4.2 and 4.3. This shows that unblanched Amaranthus hybridusleaves has a significantly (p<0.05) higher levels of oxalate and phytate content.
There was a significant (p<0.05) decrease in the oxalate and phytate content of the leaves after blanching with oxalate content of Amaranthus hybridus recording the highest decrease.
58
40
35
30
25 Unblanched Blanched
20 oncentration (mg/100g) oncentration
15 C C C
10
itamin V 5
0 Basella alba Amaranthus hybridus Vegetables
Figure.4.1: Vitamin C Content of Unblanched and Blanched Basella alba and Amaranthus hybridus Leaves.
59
2.5
2
1.5
unblanched
1 blanched Phytate (g/100g) Phytate
0.5
0 Basella alba Amaranthus hybridus Leaves
Figure 4.2. Phytate Content of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves
60
100
90
80
70
60
50
40 unblanched
blanched Oxalate (mg/100g) Oxalate 30
20
10
0 Basella alba Amaranthus hybridus Leaves
Figure 4.3: Oxalate Content of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves
61
4.3 Mineral Content of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves
The mineral content of blanched and unblanched Basella alba and Amaranthus hybridus leaves are shown in Table 4.1. The result shows that Basella alba has significantly
(p<0.05) higher content of Fe, Ca, Cu, Zn and Mg than Amaranthus hybridus leaves.
Blanching significantly (p<0.05) decreased the level of all the minerals analysed in both the leaves.
4.4 Speciation Pattern of Iron in Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves
Speciation pattern of iron in blanched and unblanchedBasella alba, and Amaranthus hybridusleaves is presented in Table 4.2. The result shows that the highest form of iron present in the leaves is the residual forms of iron with the exception of Amaranthus hybridus having exchangeable iron as the highest. However there was decrease in the exchangeable iron in both the vegetables after blanching. In-vitro bioavailable iron was higher in Amaranthus hybridus than Basella albawhich also decreased after blanching.
62
63
Table 4.1:Some Mineral ContentofBlanched and UnblanchedBasella albaand Amaranthus hybridusLeaves
Mineral elements (mg/100g)
Leaves
Fe Ca Mg Cu Zn
Unblanched Basella alba 43.05 ±1.00d 257.33 ± 2.52e 1562.45 ± 15.64d 1.50 ± 0.05d 20.89 ± 1.73d
Blanched Basella alba 25.23 ± 1.75b 201.70 ±3.24a 871.50 ±7.36a 0.60 ± 0.08b 5.63 ± 0.06a
Unblanched Amaranthus 30.91 ± 1.01c 238.19 ±2.07b 1308.78 ±7.13c 0.79 ± 0.05c 11.61 ± 0.44c hybridus
64
Blanched Amaranthus 5.98 ± 0.01a 200.66 ±6.42a 1217.08 ±2.55b 0.39 ± 0.03a 7.57 ±0.48b hybridus
Values are mean ± S.D of three determinations. Values with different superscript down the column differ significantly (p<0.05)
65
Table 4.2 Iron Speciation Pattern of Blanched and Unblanched Basella alba and Amaranthus hybridusLeaves
Iron speciation pattern (mg/100g)
EXC RED OXI RES S.O.F BAV % BAV
Leaves
Unblanched Basella 8.56 0.80 3.02 18.54 30.92 12.38 40.00 alba
Blanched Basella 5.68 3.29 0.9 3.99 13.86 9.87 71.21 alba
Unblanched 26.21 9.82 4.37 12.49 52.89 40.40 76.38 Amaranthus hybridus
Blanched 2.86 3.25 9.14 12.29 27.54 15.26 55.37 Amaranthus hybridus
EXC: Exchangeable, RED: Reducible, OXI: Oxidizable, RES: Residual, S.O.F: Sum of Fraction, BAV: Bioavailable
66
4.5 Weight Change of Iron Deficient Rats Supplemented with Blanched and UnblanchedBasella albaand Amaranthus hybridusLeaves
Weight change of iron deficient rats during supplementation with blanched and unblanched Basella alba and Amaranthus hybridus leavesis shown in Figure 4.4. The results show that there was no significant (p>0.05) difference in weight change of the rats at day7 and 14 compared to normal control. However, the anaemic control group recorded a significant (p<0.05) decrease in weight changeat day 21, with no significant
(p>0.05) difference recorded in other anaemic treated groups when compared to the normal control.
4.6 Effect of Diet Supplemented with Blanched and Unblanched Basella alba and Amaranthus hybridus leaves on Serum Ferritin Concentration.
The serum concentration of ferritin in iron deficient rats supplemented with blanched and unblanched Basella alba and Amaranthus hybridus leaves is presented in Figure 4.5. The result shows that there is a significant (p<0.05) decrease in the serum ferritin concentration of the anaemic control when compared with the normal control group. Also in the supplemented groups, there is a significant (p<0.05) decrease in the serum ferritin concentration except in unblanched Amaranthus hybridus group which recorded no significant (p<0.05) decrease.
4.7 Haemoglobin Concentration of Iron Deficient Rats Supplemented withBlanched and UnblanchedBasella albaand Amaranthus hybridusLeaves
67
The haemoglobin (Hb) concentration of iron deficient rats during supplementation with blanched and unblanchedBasella albaand Amaranthus hybridusleavesis shown in Figure
4.6. At day 0, the haemoglobin concentration of rats in all the treated groups were significantly (p<0.05) lowerthan the normal control group, but there was no significantly
(p>0.05) difference between the haemoglobin concentration of the anaemic control and the rest of the treatment groups.
At day 7 there was a significant (p<0.05) decrease in Hb of anaemic control compared to the normal control. Rats receiving Basella alba recorded a significant (p<0.05) decrease in Hb while no significant difference (p>0.05) was recorded in the groups supplemented with Amaranthus hybridus compared to the normal control.
At day 21 there was a significant (p<0.05) decrease in the haemoglobin concentration when anaemic controlwas compared to the normal control group. However, no significant
(p<0.05) difference in Hb was recorded in the supplemented groups compared to the normal control and there was significant (p<0.05) increase compared to the anaemic control.
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69
70
60
50 NRC
40 ARC AR+ISD AR+IDD+%10 UBA 30
AR+FeDD+%10 BBA Weight Change Weight (g) AR+IDD+10% UAH 20 AR+IDD+10% BAH AR+ IDD+ FeSO4 10
0 7 14 21 Days
Figure 4.4: Effect of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves Supplemented Diet on Weight Change of Iron Deficient Rat.
NRC- Normal control: Normal Rats on Iron Sufficient Diet, ARC-Anaemic Rats Control: Anaemic Rats on Iron Deficient Diet, AR+ISD- Anaemic Rats on Iron Sufficient Diet, AR+IDD+10% UBA- Anaemic Rats on Iron Deficient Diet + 10% Unblanched B.alba, AR+IDD+10% BBA-Anaemic Rats on Iron Deficient Diet + 10% Blanched B.alba, AR+IDD+10% UAH-Anaemic Rats on Iron Deficient Diet + 10% Unblanched A.hybridus, AR+IDD+10% BAH-Anaemic Rats on Iron Deficient Diet + 10% Blanched
A.hybridus, AR+IDD+ FeSO4-Anaemic Rats on Iron Deficient Diet + 2.78mg/kg Ferrous Sulphate.
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250
200
150 NRC ARC AR+ISD
100 AR+IDD+ 10% UBA AR+IDD+10% BBA AR+IDD+10% UAH
AR+IDD+10% BAH Ferritin Concentration (ng/ml) Concentration Ferritin 50 AR+IDD+FeSO4
0
Groups Figure 4.5. Effect of Blanched and Unblanched Basella alba and Amaranthus hybridus Supplemented Diet on Serum Ferritin of Iron Deficient Anaemic Rats.
NRC- Normal control: Normal Rats on Iron Sufficient Diet, ARC-Anaemic Rats Control: Anaemic Rats on Iron Deficient Diet, AR+ISD- Anaemic Rats on Iron Sufficient Diet, AR+IDD+10% UBA- Anaemic Rats on Iron Deficient Diet + 10% Unblanched B.alba, AR+IDD+10% BBA-Anaemic Rats on Iron Deficient Diet + 10% Blanched B.alba, AR+IDD+10% UAH-Anaemic Rats on Iron Deficient Diet + 10% Unblanched A.hybridus, AR+IDD+10% BAH-Anaemic Rats on Iron Deficient Diet + 10% Blanched
A.hybridus, AR+IDD+ FeSO4-Anaemic Rats on Iron Deficient Diet + 2.78mg/kg Ferrous Sulphate.
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14
13
NRC 12 ARC 11 AR+ISD AR+IDD+%10 UBA 10 AR+FeDD+%10 BBA
HaemoglobinConcentration (g/dl) 9 AR+IDD+10% UAH AR+IDD+10% BAH 8 AR+ IDD+ FeSO4
7 0 7 14 21 DAYS
Figure 4.6Effect of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves Supplemented Diet on Haemoglobin Concentration of Iron Deficient Rats.
NRC- Normal control: Normal Rats on Iron Sufficient Diet, ARC-Anaemic Rats Control: Anaemic Rats on Iron Deficient Diet, AR+ISD- Anaemic Rats on Iron Sufficient Diet, AR+IDD+10% UBA- Anaemic Rats on Iron Deficient Diet + 10% Unblanched B.alba, AR+IDD+10% BBA-Anaemic Rats on Iron Deficient Diet + 10% Blanched B.alba, AR+IDD+10% UAH-Anaemic Rats on Iron Deficient Diet + 10% Unblanched A.hybridus, AR+IDD+10% BAH-Anaemic Rats on Iron Deficient Diet + 10% Blanched
A.hybridus, AR+IDD+ FeSO4-Anaemic Rats on Iron Deficient Diet + 2.78mg/kg Ferrous Sulphate.
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4.8 Effect of Blanched and Unblanched Basella alba and Amaranthus hybridus on Some Haematological Parameters.
The haematological parameters of iron deficient rats fed with diet supplemented with blanched and unblanched Basella alba and Amaranthus hybridus leaves at the end of 21 days is presented in Table 4.3. The results show that there was significant (p<0.05) decrease in packed cell volume (PCV), Hb, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration
(MCHC), no significant (p>0.05) difference in red blood cell (RBC) count and white blood cell (WBC) count of the anaemic control when compared to the normal control. In the supplemented groups, there was no significant (p>0.05) difference in RBC, PCV, Hb,
MCHC compared to the normal control with the exceptions of RBC for unblanched
Amaranthus hybriduswhich recorded significant (p<0.05) increase and PCV for unblanched Basella alba which recorded a significant (p<0.05) decrease. A significant
(p<0.05) decrease was obtained in WBC, MCH and MCV of supplemented groups compared to normal control with the exception of WBC of unblanched Basella alba which recorded no significant (p>0.05) difference.
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Table 4.3. Effect of Blanched and Unblanched Basella alba and Amaranthus hybridusLeaves Supplemented Diet on Some
GROUP RBC count WBC Count PCV Hb MCH MCV MCHC
n=6 (x106µl) (x106µl) (%) (g/dL) (pg) (fl) (g/dL)
NRC 4.12 ± 0.16a 12.37± 1.91b 39.17± 0.75b 13.14 ± 0.11bc 32.48 ± 0.68c 96.71 ± 1.37c 33.59 ± 0.26b
ARC 4.03 ± 0.25a 9.38± 0.71ab 35.00±1.73a 10.17 ± 0.15a 26.37 ± 0.56a 86.84 ± 2.34a 30.37 ± 0.41a
AR+ISD 4.33 ± 0.32a 8.54± 0.85a 38.50±1.00ab 12.85 ± 0.24bc 30.57 ± 0.38b 91.21 ± 1.27b 33.51 ± 0.15b
AR+IDD+10% UBA 4.10 ± 0.32a 11.92± 1.34b 35.17±3.37a 12.44 ± 0.95b 30.35 ± 0.90b 87.70 ± 1.60ab 34.59 ± 1.43b
AR+1DD+10% BBA 4.16 ± 0.05a 6.96± 0.89a 38.20±3.56ab 12.37 ± 0.33b 29.36 ± 0.14b 86.39 ± 1.49a 33.99 ± 0.42b
AR+1DD+10% UAH 4.80± 0.10b 6.20± 0.99a 40.67±0.58b 13.35 ± 0.72c 29.11 ± 1.47b 86.68 ± 4.26a 33.58 ± 0.47b
AR+1DD+10% BAH 4.25± 0.17a 6.10± 0.52a 38.50±3.32ab 12.47 ± 0.50b 29.37 ± 0.60b 87.20 ± 1.30ab 33.67 ± 0.47b
a a ab bc b ab b AR+IDD+ FeSO4 4.37± 0.12 8.30± 1.05 37.50±1.92 12.83± 0.32 29.44 ± 0.15 88.63 ± 0.37 33.21 ± 0.30
Haematological Parameters in Iron Deficient Rats
Values are means ± SD of three determinations. Values with different superscript down the column are significantly (P<0.05) different
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Key; NRC; Normal Rats as Control, ARC; Anaemic Rat control, AR+ISD; Anaemic Rats + Iron Sufficient Diet, AR+IDD+10% UBA; Anaemic Rat+ Iron Deficient Diet + 10% Unblanched Basella alba,AR+IDD+10% BBA; Anaemic Rat+ Iron Deficient Diet + 10% Blanched Basella alba, AR+IDD+10% UAH;Anaemic Rat+ Iron Deficient Diet + 10% Unblanched Amaranthus hybridusAR+IDD+10% BAH; Anaemic Rat+ Iron Deficient Diet + 10% Blanched Amaranthus hybridus,AR+IDD+FeSO4; AnaemicRats+ Iron Deficient Diet + 2.78mg/kg Ferrous Sulphate.
RBC; Red blood cells, WBC; White blood cells, PCV; Pack cell volume, Hb;Haemoglobin Concentration, MCH; Mean Corpuscular Haemoglobin, MCV; Mean Corpuscular Volume, MCHC; Mean Corpuscular Haemoglobin Concentration.
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CHAPTER FIVE
5.0 DISCUSSION
Vegetables have been widely used for their health promoting properties, as they have been found to be rich in vitamins, minerals and phytochemicals (Oboh et al., 2005;Slavin and
Lloyd, 2012).Report has shown that consumption of diets rich in vegetables and fruits protect the human body from chronic degenerative diseases such as cancer, hypertension and anaemia (Babalola et al., 2010). Iron is a very important mineral that is required by the body for its proper functioning.It has been found that vegetables are very important source of dietary non-haem iron(NHS, 2011; Slavin and Lloyd, 2012). The use of vegetable as a source of dietary iron has many advantage over the use of iron supplement because there is less risk of iron overload and there will be no problem with the compliance to drug regiment as the iron is consumed as part of diet (Lewis, 2014).
Vitamin C, also known as ascorbic acid is one of the most important vitamin in fruits and vegetables.It functions in the synthesis of collagen,reduction of plasma cholesterol level, inhibition of nitrosamine formation, enhancement of the immune system, and as an anti-oxidant which react with singlet oxygen and other free radicals to reduces the risk of arteriosclerosis, cardiovascular diseases and some forms of cancer (Kumar et al.,
2013b).The significantly (p>0.05) higher vitamin C content ofAmaranthus hybridus disagree with the work of Ogunlesi et al., (2010) which report that Basella alba has higher vitamin C content than Amaranthus hybridus. The significant (p<0.05) decrease in vitamin C of both vegetables after blanching can be attributed to the decomposition of vitamin C in the
77 presence of heat. Vitamin C is a heat labile and water soluble vitamin which can easily leach into the water that was used for the purpose of blanching (Babalola et al., 2010).
Phytate is a salt of phytic acid, which is a bioactive compound that is widely distributed in plant food. Due to its molecular structure, phytic acid has affinity for polyvalent cations, such as minerals and trace elements and could in this way interfere with their intestinal absorption in man and in other animals (Oboh et al., 2005). Phytate and oxalate are inhibitors of iron absorption, they interfere with the absorption of iron in the intestine by formation of complexes with the iron which makes them insoluble and poorly absorbable to the body (Noonan, 1999). At physiologically relevant pH values (pH 1.5–7), phytic acid is negatively charged, accounting for the ability of phytate to chelate mineral cations. The more phosphorylated the inositol rings, the stronger the interaction with iron and the lower the solubility. At physiological conditions, i.e., in the intestinal environment, phytate thus forms complexes with ferric iron to form monoferric phytate (Nielsen et al., 2013).
Oxalates are known to interfere with the absorption of Ca by reducing its availability due to the formation of calcium oxalate in the intestine of monogastric animals, but has little or no effect on the absorption of Zn (Liener, 1994).The significantly (p<0.05) higherphytate and oxalate content ofAmaranthus hybridus leaves can be due to quality of soil the vegetable was planted on and the state or degree of ripeness of the vegetables (Brogen and Savage,
2003). The significant (p<0.05) reduction in phytate content in all the vegetables disagrees with the earlier report by Adegunwaet al., (2011), that blanching increases the phytate content of vegetables. The reduction in these anti-nutritional factors can be as a result of
78 blanching which rupture the superficial layer of the vegetables where the concentration of the anti-nutritional factors are highest (Virginia and Swati, 2012).
Minerals are inorganic nutrients that are required by the body in small amount from less than
1 to 2500mg per day depending on the minerals. Minerals are necessary for the maintenance of certain physico-chemical processes which are essential to life. Although they yield no energy, but they play important roles in different metabolic processes (Soetan et al., 2010).
Fruits and vegetables are widely recommended for their health promoting properties, they have high concentration of minerals especially electrolytes (Slavin and Lloyd, 2012).Basella alba has significantly (p<0.05) higher minerals content than Amaranthus hybridus which can be due to difference in soil composition and rate of mineral intake by the individual plant (Asaolu and Asaolu, 2010). The significant (p<0.05) decrease in the mineral content of both the vegetables after blanching agrees with the finding of De Corcuera, (2004)which suggested that depending on the type and duration of blanching it can affect the color, texture and the nutritional content of vegetables. Minerals and vitamins leach out of the vegetables during blanching, especially water blanching (Kawashima and Valente-Soares,
2005).
Metal bioavailability in sediments depends strongly on the mineralogical and chemical forms they occurs (Baeyens et al., 2003), therefore its very imperative to study different forms of metals mobility and bioavailability rather than the total concentration in order to obtain the indication of the bioavailability of the metal (Fan et al., 2002; Nadaska et al.,
2009). The speciation pattern of the vegetables was carried out so as to predict the impact of
79 the iron metal in the body, factors such as soil, rainfall, vicinity of industry and extensive agricultural activity influence the level of bioavailable elements in plants (Konieczyński and
Wesołowski, 2007). However, Li and Deng (2004) also pointed out that the value of bioavailable fraction obtained depends on the applied type of extraction solvent, plant part and the botanical species of a plant.High amount of residual iron found in Basella alba which agrees with the findings of Majolagbeet al., (2013) and Owolabi,et al., (2015), whoreported that iron is strongly bonded to the plant tissues. The percentage bioavailability of Amaranthus hybridus was higher due to the high concentration of exchangeable iron in the leaves. The percentage bioavailability of Amaranthus hybridus decreased after blanching due to decrease in residual iron concentration while it increased in Basella alba due to reduction of residual iron.
The significant (p< 0.05) decrease in the weight change of the anaemic control group suggest that iron is important for animal growth, as described by Yi Ninget al., (2002), who reported that iron deficiency impair weight gain and reduce final body weight. Thedecrease in weight is in agreement with the work of Beard et al., (1996), Huh et al., (1999), Soltan,
(2013) and Okolo et al., (2015), they reported that administration of iron deficient diet caused reduction in the final body weight, body weight gain, and food consumption.
Ferritin is considered the most important indicator of iron status and a central parameter for determination of therapeutic response (Mei et al., 2005; O‘Meara et al., 2011). Serum ferritin is the first parameter to reflect iron deficiency even when the haemoglobin
80 concentration, haematocrit level and serum iron are normal (Short and Domagalski, 2013).
Therefore serum ferritin is an important biochemical parameter for monitoring iron deficiency in the absence of inflammation and infection and also it can be used to differentiate between anaemia of chronic disease with iron deficiency (Walker et al., 2010).
Serum ferritin is directly implicated in potentially devastating diseases, reduction is an indication of depletion of iron store which is a marker for iron deficiency anaemia (Mei et al., 2005). At the end of 21 days of diet supplementation, the serum ferritin concentration of the iron deficient anaemic control group was found to be the lowest which can be due to the depletion of iron stores (O‘Meara et al.,2011). The groups that received iron deficient diet supplemented with 10% Amaranthus hybridus had the highest increase in the level of serum ferritin level which can be attributed to high levels of vitamin C and low level of Ca in
Amaranthus hybridus leaves. Vitamin C increase the absorption of iron in the intestine by reducing Fe 3+ to Fe 2+, and Ca chelates ironthereby interfering with absorption (Sharp and
Srai, 2007; Nielsen et al., 2013).
Haematological parameters have been associated with health indices and are of diagnostic significance in routine clinical evaluation of state of health. It is an established fact that chronic diseases affect the blood cells adversely (Saliu et al., 2012). Haemoglobin concentration level is used in diagnosis of iron deficiency anaemia. This is because haemoglobin is the iron-containing protein found in red blood cells that allows the red blood cells to function as the oxygen transport system to the tissues of the body. The haemoglobin concentration of the animals on iron deficient diet met the cut-off value
(<11g/dl) after 14 weeks of feeding, this is in contrast with the work of Okolo et al., (2015)
81 who reported that iron deficiency anaemia was induced by the eighth week of feeding.
This might be because the diet used in this study was not completely deficient in iron also or the amount of iron stores in the rats could also delay the induction. The haemoglobin concentrations for all the rats except those in the Iron sufficient control group were significantly (p<0.05) low, this agrees with Okolo et al., (2015) and Joshi and Mathur
(2009). Decreased haemoglobin might be due to the effect of a shortage of iron available to the erythroid precursors in the bone marrow for haemoglobin synthesis
(Cook, 2005).
At the end of 21 days of supplementation period, the significant (p<0.05) increase in the haemoglobinconcentration level of all the vegetable supplemented groups when compared with the anaemic control group might be due to the availability of iron from the vegetable been a good source of dietary iron (Sheela et al., 2004).
Next to haemoglobin as a parameter for diagnosis of iron deficiency is the haematocrit (Ht) or packed cell volume (PCV) which is a measure of the portion of the blood volume made up by red blood cells. Mean corpuscular volume (MCV) and Mean corpuscular haemoglobin (MCH) are two good red blood cell indices that are used in the diagnosis of iron deficiency anaemia. A significant (p<0.05) decrease in the MCH and no significant
(p>0.05)difference in the MCV of the anaemic control when compared with diet supplemented groups agrees with the findings of Jolobe, (2012),who reported that MCH appears to be superior to MCV in the distinction of iron deficiency and other types or forms
82 of anaemia. Also according to the study conducted by Francis et al., (2005), high proportion of patient with iron deficiency anaemia had a low MCH compared with low MCV, and also when the Hb level and the serum ferritin level of the patient is low MCH correlate better iron deficiency anaemia than MCV.
The unblanched Basella alba group recorded a significant (p<0.05) increase in the WBC concentration which is in line with the findings of Bamidele et al., (2010) who reported thatBasella alba increased the level of haematological parameters in an albino rat.The significant (p<0.05) decrease in the Hb, PCV, MCHC, MCH and MCV in the anaemic control rats is due to the induction of anaemia. During the development of negative iron balance in the body with no mobilization from iron stores, there will be immediate impairment in the production of haemoglobin with the resulting decrease in haemoglobin and different red blood cell indices such as mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) (Hallberg, 1995;Butensky et al., 2008).
CHAPTER SIX
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS
6. 1 SUMMARY
From this study, Amaranthus hybridushas a high amount of vitamin C (35.67 ± 0.15 mg/100g) which is significantly (p<0.05) higher than that of Basella alba (30.27± 0.25 mg/100g). Blanching significantly (p<0.05) decreased the vitamin C content of Amaranthus hybridus and Basella albaby 42.81 and 64.42 %respectively.
83
The vegetables were also found to be abundant in phytate and oxalate, with Amaranthus hybridus (2.06 ±0.13mg/100g) having significantly (p<0.05) higher phytate content than
Basella alba(1.74±0.07g/100g), also Amaranthus hybridus (89.75±mg/100g) has significantly (p<0.05) higher amount of oxalate than Basella alba(72.37±mg/100g). Both phytate and oxalate were decreased significantly (p< 0.05) by blanching.
Basella alba was found to have high mineral content of the elements analysed when compared with Amaranthus hybridus. It has higher content of Mg (1562.45 ± 15.64), Ca
(257.33 ± 2.52), Fe (43.05 ±1.00) and Zn (20.89 ± 1.73) all in mg/100g. Blanching significantly (p<0.05) decreased all the minerals analysed in both vegetables.
Unblanched Amaranthus hybridus has the highest In-vitro bioavailability of 76.38% over the blanched Amaranthus hybridus (55.37%), unblanched Basella alba (40.00%) and blanched
Basella alba (71.21%). The most common form of iron in the blanched Amaranthus hybridus and unblanched Basella alba is the residual form, while the exchangeable form of iron was highest in unblanched Amaranthus hybridus and blanched Basella alba.
There was no significant (p>0.05) difference in the serum ferritin level when the normal rats are compared with the groups that received iron deficient diet supplemented with the vegetables, also there was no significant (p>0.05) difference in the serum ferritin level when the test groups are compared to each other with the group that was given unblanched
Amaranthus hybridus (138.40 ±19.90 ng/mL) having the highest serum ferritin level.
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Diet supplementation with unblanched Amaranthus hybridus significantly (p<0.05) increased RBC (11.97 ± 1.34 x 106µl) and PCV (40.67 ± 0.58 %) when compared with the anaemic control group, blanched and unblanched Basella alba groups and blanched
Amaranthus hybridus group. The group that received the unblanched Basella alba had a significant (p<0.05) increase in the WBC when compared with the diet supplemented groups and the anaemic control group.There was no significant (p>0.05) difference in the MCH and the MCHC when the diet supplemented groups are compared with the normal rats. Also there is a significant (p<0.05) decrease in the MCV of the diet supplemented group when compared with the normal rats.
6.2 CONCLUSION
The results suggest increased bioavailability of iron from Basella alba and Amaranthus hybridus supplemented diet in iron deficient anaemic albino rats with unblanched vegetables exhibiting greater potentials than the blanched vegetables.
6.3 RECOMMENDATIONS
The effect of processing other than heat processing methods on bioavailability of
iron in Basella alba and Amaranthus hybridus should be carried out.
Increase consumption of these vegetables by people who are at the risk to iron
deficiency (under five children, pregnant and lactating mother‘s women of
reproductive age) should be encourage.
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APPENDICES:
Appendix 1
FLOW CHART FOR EXPERIMENTAL DESIGN
COLLECTION AND IDENTIFICATION OF LEAVES
AIR DRYING OF LEAVES BLANCHING AND OVEN DRYING OF LEAVES
FORMULATION OF IRON DEFICIENT AND IRON SUFFICIENT DIET
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ACCLIMATIZATION OF ANIMALS
INDUCTION OF ANAEMIA
FORMULATION OF SUPPLEMENTAL DIETS
TEST ON ANIMALS
BODY WEIGHT
MEASUREMENT BIOCHEMICAL ASSAY
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HAEMATOLOGICALAS SAY
STATISTICAL ANALYSIS
Appendix 2
Content of Vitamin and Mineral Mix (Vitalyte Extra TM)
Specification per 1000g
Nutrient Unit/1000g
Vitamin A 15,000,000 IU
Vitamin D3 4, 400,000 IU
Vitamin E 2,500 IU
Vitamin K 4,350 mg
Vitamin B2 4,350 mg
Vitamin B6 2,350 mg
Vitamin B12 11,350 mg
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Vitamin C 1000 mg
Nicotinamide 16,700 mg
Calcium Pantothenate 5,350 mg
Potassium Chloride 87,000 mg
Sodium Sulphate 212,000 mg
Sodium Chloride 50,000 mg
Magnesium Sulphate 12,000 mg
Copper Sulphate 12,000 mg
Zinc Sulphate 12,000 mg
Manganese Sulphate 12,000 mg
Lysine Hydrochloride 15,000 mg
Methionine 10,000 mg
Expicients Q.S 1,000 g
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Appendix 3
0.14
0.12
0.1
0.08
Absorbance 0.06
0.04
0.02
0 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration (mg/ml)
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Standard Curve for Vitamin C
Appendix 4
115
0.5
0.45
0.4
0.35
0.3
0.25 Absorbance 0.2
0.15
0.1
0.05
0 0 1 2 3 4 5 6 7 8
Concentration (mg/1000g)
Standard Curve for Fe
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Appendix 5
117
0.18
0.16
0.14
0.12
0.1
0.08 Absorbance 0.06
0.04
0.02
0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 -0.02 Concentration (mg/1000g)
Standard Curve for Zn
118
Appendix 6
119
0.3
0.25
0.2
0.15 Absorbance
0.1
0.05
0 0 1 2 3 4 5 6 7 8 9
Concentration (mg/1000g)
Standard Curve for Cu
120
Appendix 7
0.4
0.35
0.3
0.25
0.2 Absorbance 0.15
0.1
0.05
0 0 0.2 0.4 0.6 0.8 1 1.2 Concentration (mg/1000g)
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Standard Curve for Magnesium
Appendix 8
122
0.45
0.4
0.35
0.3
0.25
0.2 Absorbance 0.15
0.1
0.05
0 0 10 20 30 40 50 60 -0.05 Concentration (mg/1000g)
Standard Curve for Ca
123
Appendix 9
3.5
3
2.5
2
1.5 Optical Density Optical
1
0.5
0 0 50 100 150 200 250 300 350 400 450 Concentration (ng/ml)
124
Standard Curve for Serum Ferritin
Appendix 10
Vitamin C content in Blanched and Unblanched Basella alba and Amaranthus hybridusLeaves
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Leaves Vitamin C(mg/100g)
Unblanched Basella alba 30.17 ± 4.19c
Blanched Basella alba 10.77 ± 2.11a
Unblanched Amaranthus hybridus 35.67 ± 2.85d
Blanched Amaranthus hybridus 20.40 ± 0.20b
Values are mean± SD of three determinations. Means with different superscript down the column differ significantly (p<0.05).
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Appendix 11
Antinutrients Content of Unblanched and Blanched Basella alba and Amaranthus hybridusLeaves
Leaves ANTINUTRIENT
Phytate(g/100g) Oxalate(mg/100g)
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Basella alba 1.74 ± 0.07b72.37 ± 2.15c
Basella alba (Blanched) 1.17 ± 0.16a 58.72 ± 1.18b
Amaranthus hybridus 2.06 ± 0.13c 89.75 ±0.57dAmaranthus hybridus(Blanched)
1.60 ±0.18b 49.05 ±0.18a
Values are mean± SD of three determinations. Means with different superscript down the column differ significantly (p<0.05).
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Appendix 12
Effect of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves Supplemented Diet on Weight Change of Iron Deficient Rat.
GROUP Day 0(g) Day 7(g) Day 21(g)
n=6
NRC 12.00 ± 43.67 ± 53.67 ±
ARC 10.67 ± 15.67 ± 26.00 ±
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AR+ISD 24.00 ± 34.67 ± 38.00 ±
AR+IDD+10% 27.33 ± 36.33 ± 45.67 ± UBA
AR+1DD+10% 20.33 ± 31.33 ± 44.67 ± BBA
AR+1DD+10% 26.33 ± 38.33 ± 45.33 ± UAH
AR+1DD+10% 16.33 ± 27.67 ± 39.33 ± BAH
AR+IDD+FeSO4 21.33 ± 31.33 ± 40.00 ±
Values are mean ± S.D of three determinations. Values with different superscript down the column and subscript across the period differ significantly (p<0.05)
NRC- Normal control: Normal Rats on Iron Sufficient Diet, ARC-Anaemic Rats Control: Anaemic Rats on Iron Deficient Diet, AR+ISD- Anaemic Rats on Iron Sufficient Diet, AR+IDD+10% UBA- Anaemic Rats on Iron Deficient Diet + 10% Unblanched B.alba, AR+IDD+10% BBA- Anaemic Rats on Iron Deficient Diet + 10% Blanched B.alba, AR+IDD+10% UAH- Anaemic Rats on Iron Deficient Diet + 10% Unblanched A.hybridus, AR+IDD+10% BAH- Anaemic Rats on Iron Deficient Diet + 10% Blanched A.hybridus,
AR+IDD+ FeSO4- Anaemic Rats on Iron Deficient Diet + 2.78mg/kg Ferrous Sulphate.
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Appendix 13
Effect of Blanched and Unblanched Basella alba and Amaranthus hybridus Leaves Supplemented Diet on Haemoglobin Concentration of Iron Deficient Rats.
GROUP DAY 0 (g/dL) DAY 7 (g/dL) DAY 21 (g/dL)
n=6
NRC 12.96 ± 12.60 ± 13.14 ±
ARC 9.43 ± 9.60 ± 10.17 ±
AR+ISD 9.23 ± 11.13 ± 12.85 ±
AR+IDD+10% 8.82 ± 10.72 ± 12.44 ± UBA
AR+1DD+10% 8.93 ± 10.30 ± 12.38 ± BBA
AR+1DD+10% 9.38± 11.10 ± 13.35 ± UAH
AR+1DD+10% 9.13± 10.88 ± 12.48 ± BAH
AR+IDD+FeSO4 8.73± 11.00 ± 12.83±
131
Values are mean ± S.D of three determinations. Values with different superscript down the column and subscript across the period differ significantly (p<0.05)
NRC- Normal control: Normal Rats on Iron Sufficient Diet, ARC-Anaemic Rats Control: Anaemic Rats on Iron Deficient Diet, AR+ISD- Anaemic Rats on Iron Sufficient Diet, AR+IDD+10% UBA- Anaemic Rats on Iron Deficient Diet + 10% Unblanched B.alba, AR+IDD+10% BBA- Anaemic Rats on Iron Deficient Diet + 10% Blanched B.alba, AR+IDD+10% UAH- Anaemic Rats on Iron Deficient Diet + 10% Unblanched A.hybridus, AR+IDD+10% BAH- Anaemic Rats on Iron Deficient Diet + 10% Blanched
A.hybridus, AR+IDD+ FeSO4- Anaemic Rats on Iron Deficient Diet + 2.78mg/kg Ferrous Sulphate.
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Appendix 14
Effect of Basella alba and Amaranthus hybridus Leaves Supplementation on Serum Ferritin Concentration of Iron Deficient Rats
Group Ferritin concentration (ng/ml)
N=6
NRC 170.00 ± 33.01c
ARC 96.67 ± 20.81a
AR+FSD 141 ± 17.00bc
AR+FDD+ 10% Unblanched 121.33 ± 20.50ab B.alba
A+FDD+10% Blanched B.alba 124.00 ± 27.78ab
AR+FDD+10% Unblanched A. 138.40 ± 19.90bc Hybridus
AR+FDD+10% Blanched A. 116.20 ± 11.45ab Hybridus
bc AR+ IDD+ FeSO4 139.50 ± 20.60
Values with different superscript down the column differ significantly (p<0.05)
NRC- Normal control: Normal Rats on Iron Sufficient Diet, ARC-Anaemic Rats Control: Anaemic Rats on Iron Deficient Diet, AR+ISD- Anaemic Rats on Iron Sufficient Diet, AR+IDD+10% UBA- Anaemic Rats on Iron Deficient Diet + 10% Unblanched B.alba, AR+IDD+10% BBA- Anaemic Rats on Iron Deficient Diet + 10% Blanched B.alba, AR+IDD+10% UAH- Anaemic Rats on Iron Deficient Diet + 10% Unblanched A.hybridus, AR+IDD+10% BAH- Anaemic Rats on Iron Deficient Diet + 10% Blanched
133
A.hybridus, AR+IDD+ FeSO4- Anaemic Rats on Iron Deficient Diet + 2.78mg/kg Ferrous Sulphate.
134
Appendix 15
Preparation of Reagents Used for Speciation Pattern Analysis
Magnesium chloride (1M) was prepared by weighing 20.33g of the salt in a 100ml volumetric flask , little deionized water was added to dissolve it after which more water was added to make it up to the 100ml mark.
Sodium acetate (1M ) was prepared by weighing 8.203g of the salt in a 100ml volumetric flask, little deionized water was added to dissolve it after which more water was added to make it up to the 100ml mark.
Hydroxylamine hydrochloride (1M ) was prepared by weighing 17.37g into a 250ml volumetric flask , little deionized water was added to dissolve it after which more water was added to make it up to the 250ml mark.
Hydroxylamine hydrochloride (0.25M) was obtained by taking 14.39ml from the already prepared 1M Hydroxylamine hydrochloride
Hydrochloric acid (12M) was prepared by measuring 248.70ml of the acid and deionized water was added by the sides to make the volume up to the 250ml mark.
Aqua Regia Solution was prepared by measuring 1.2 liters of distilled water into a 2 litres volumetric flask 400ml concentration HCl and 133ml of 70% Nitric acid was added carefully, and was diluted to 2 liters.
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Appendix 16
Preparation of standard drug stock solution
A stock solution of FeSO4 with a concentration of 19.5 mg per ml was prepared by dissolving
3 tablets of the drug and made up to 10ml with deionized water. 2.86ml was taken from the stock solution and made up to 20ml with deionized water to prepare a concentration of 2.79 mg. The drug was administered to the rats at a dose of 2.78mg/kg
136
Appendix 17
Mineral content of the control and supplemented diets
DIETS MINERALS (mg/80g)
Fe Zn Cu Ca
Fe def. diet 13.92 3.06 10.20 22.02
Fe suf. Diet 286.9 2.86 4.58 40.58
Fe def. diet + 10% Unblanched B. alba 17.52 4.73 10.32 42.61
Fe def. diet + 10% Blanched B. alba 15.94 3.51 10.25 38.16
Fe def. diet + 10% Unblanched A.hybridus 16.39 3.99 10.26 41.08
Fe def. diet + 10% Blanched A.hybridus 14.40 3.67 10.23 38.07
Fe def: Iron deficient, Fe suf: Iron sufficient
137