MICRONUTRIENT CONTENT AND EFFECTS OF JATROPHA CURCAS AND BRILLANTASIA NITENS LEAVES EXTRACTS IN ANAEMIA INDUCED ADULT MALE ALBINO RATS

TABLE OF CONTENTS

Title page ……………………………………………………………………………….      I

Approval page …………………………………………………………..        II

Certification page ………………………………………………………..…..      III

Dedication ………………………………………………………………………………     IV

Acknowledgement …………………………………………………………     V

List of Tables……………………………………………………………………………….    X

List of Figures………………………………………………………………..     XI

List of Plates……………………………………………………………………………..   XII

Abstract …………………………………………………………………………………    XIII

CHAPTER ONE: INTRODUCTION

1.1       Background to the Study………………………………………           1

1.2       Statement of the Problem……………………………………       3

1.3       Objective of the Study……………………………………………       5

1.4       Significant of the Study…………………………………………     6

CHAPTER TWO: LITERATURE REVIEW

2.1      Malnutrition …………………………………………………….……… 7

2.1.2 Agriculture and Nutrition………………………………………………………..         7

2.1.3   Vegetables …….……………………………………………….           8

2.1.4 Health Benefit of Vegetable……………………………      9

2.2      Nutritive Value of Green Leafy Vegetables………………..   10

2.2.1   Moisture Content…………………………………………………     10

2.2.2   Carbohydrate ……………………………………………………….     10

2.2.3   Proteins………………………………………………………………    11

2.2.4   Fats………………………………………………………………..    11

2.2.5   Ash .……………………………………………………………………………….     11

2.2.6   Vitamins ……………………………………..……………..      12

2.2.6.1 Vitamin A…………………………………………….…………..    12

2.2.6.2 Vitamin B₆ …………………………………………….………    13

2.2.6.3 Folic acid…………………………………………………….…….   13

2.2.6.4 Ascorbic acid…………………………………………….………..   13

2.2.6.5 Vitamin E…………………………………………………….……   14

2.2.7    Mineral……………………………………………………………     14

2.2.7.1 Copper ………………………………………………………   15

2.2.7.2 Iron (Fe)………………………………………………………      15

2.2.7.3 Zinc (Zn)…………………………………………………………….    15

2.2.7.4 Manganese (Mn)…………………………………………………….    16

2.2.7.5 Phosphorus (P)………

2.2.7.5 Calcium (Ca)………………………………………………..     16

2.2.7.6 Potassium (K)………………………………………………     17

2.2.8    Factors that Affect Consumption of Fruits and Vegetables…… 17

2.2.8.1 Household Income………………………………………………..        17

2.2.8.2 Availability………………………………………………………     18

2.2.8.3 Consumer Preferences…………………………………        18

2.2.8.4 Culture……………………………………………………………    19

2.2.8.5 Lack of Information (Nutrition Education)…………………….    19

2.2.9    Vegetables in the Management of Anaemia……………………..      19

2.3       Jatropha curcas………………………………………………..     20

2.3.1    Cultivation of Jatropha curcas……………………………….       20

2.3.2    Food Uses and Medicinal Values of Jatropha curcas.……………..  21

2.3.4    Nutrient Composition of Jatropha curcus………………..  22

2.4       Toxicity of Jatropha curcas…………………………….         23

2.5       Brillantaisia nitens ……………………………………      23

2.5.1    Food Uses/Medicine Values of Brillantaisia nitens…………………     24

2.5.2    Nutrient Composition of Brillantaisia nitens………………  25

2.6       Phytochemical ………………………………………………..      25

2.6.1    Flavonoids…………………………………………………………..    27

2.6.2    Alkaloids…………………………………………………………….    27

2.6.3.   Saponins…………………………………………………………      28

2.7.      Anti-Nutrients…………………………………………………     28

2.7.1    Oxalate………………………………………………………………    29

2.7.2    Phytate …………………………………………………………..     29

2.7.3    Tannins……………………………………………………………..      29

2.8       Haematological Indices…………………………..……………30

2.8.1    Hemoglobin. …………………….……………………………….    30

2.8.2    Red Blood Cell…………………….………………………………  31

2.8.3    White Blood Cell ………………….…………………………..    31

2.8.4    Packed Cell Volume……………………….……………………….    32

2.8.5    Mean Corpuscular Volume (MCV)………………………. 33

2.8.6    Mean Corpuscular Hemoglobin (MCH)….……………………    33

2.8.7    Mean Corpuscular Hemoglobin Concentration (MCHC)……… 33

2.9       Liver Function Test ……………………………………………    34

2.9.1    Alanine Amino Transferase (ALT)…………………     34

2.9.2    Aspartate Amino Transferase (AST)…………………..    34

2.9.3   Alkaline Phosphatase (ALP)……………………………..       34

2.10 Micronutrients……………………………………………          35

2.10.1 Micronutrient Deficiency ……………………………..      37

2.11     Anaemia   ………………………………………………………..     38

2.11.0  Types of Anaemia……………………………………………..     39                                                                                                                                                         2.11.1  Iron Deficiency Anaemia   ……………………………     39

2.11.1.2 Signs and Symptoms of Anaemia………………………..       40

2.11.2  Haemolytic Anaemia …………………………….    41

2.11.3 Pernicious Anaemia…………………………………………………………  42

2.11.4  Aplastic Anaemia …………………………………………………   42

2.11.5  Megaloblastic Anemia…………………………………    43

2.11.6  Sickle Cell Anaemia……………………………………………………    43

2.12.1  Anemia Caused by Blood Loss …………………………… 44

2.12     Aetiology of Anemia…………………………………………………..    44

2.12.3  Anemia Caused by Decreased or Faulty Red Blood Cells………… 44

2.12.4  Diseases and Disease Treatments……………………………..     45

2.12.5  High Rates of Red Blood Cell Destruction………………..      45

2.12.6  Gastrointestinal Blood Loss…………………………………….  46

2.13     Consequences of Anaemia …………………………………. 46

2.14     Effect of Leafy Vegetables and their Extracts on Anaemia……. 48

2:15     Cyclophosphamide ………………………………………………….   49

CHAPTER THREE: MATERIALS AND METHODS

3.1       Study Design…………………………………………………………  50

3.1.1    Collection of Samples……………………………………………………     50

3.1.2    Preparation of Samples………………………………………   50

3.2       Chemical Analysis. …………………………………………………….   51

3.2.1    Proximate Analysis…………………………………………………….    51

3.2.1.1 Determination of Moisture Content……………………………..     51

3.2.1.2 Determination of Ash……………………………………………..   52

3.2.1.3 Determination of Crude Fat……………………………………………………   52

3.2.1.5 Determination of Crude Protein……………………………..            ……..    53

3.2.1.6 Determination of Carbohydrate……………………………………………..   53

3.3       Mineral Determination ……………………………………….      54

3.3.1    Iron Determination………………………………………………     54

3.3.2    Zinc Determination…………………………………………………     54

3.3.3    Copper  ………………………………………………………………      55

3.4       Vitamin Determination…………………………………       55

3.4.1    Provitamin A (Beta-carotenoid)…………………………………     55

3.4.2    Vitamin C………………………………………………………………………       55

3.4.3    Vitamin E…………………………………………………………        55

3.4.4    Vitamin B₆………………………………………………….     56

3.4.6    Vitamin B₉  ……………………………………………………        56

3.5.      Anti nutrient Determination………………………………….      57

3.5.1    Tannins………………………………………………        57

3.5.2 Phytates………………………………………………………….        57

3.5.3    Oxalate …………………………………………………………………………    ……..    57

3.6       Phytochemical Determination……………………………….        58

3.6.1    Saponins…………………………………………………………..      58

3.6.2 Flavonoid …………………………………………………………           59

3.6.3 Alkaloids………………………………………………………………………        …….   59

3.7       Rat Study…………………………………………………………..      60

3.7.1    Animal Source and Housing …………………………      60

3.7.2    Feeding of the Rats……………………………………………    60

3.7.3    Preparation of Vegetable Extract for the Rat Study………………… 61

3.7.4    Induction of Anaemia…………………………………………….    61

3.7.5    Blood Samples Collection………………………………………..    62

3.8       Haematological Determination……………………………….      62

3.8.1    Determination of Packed Cell Volume…………………………   62

3.8.2    Determination of Hemoglobin (Hb) Concentration……………..      63

3.8.3    Determination of Red Blood Count (RBC)………………   64

3.8.4    Determination of White Blood Cells Counts (WBCs)…………… 65

3.8.5    Mean Corpusular Volume (MCV)…………………………   66

3.8.6    Mean Corpuscular Hemoglobin (MCH)……………………………. 66

3.8.7    Mean Corpuscular Hemoglobin Concentration (MCHC) ………… 66

3.9       Liver Function Test ………………………………………………    67

3.9.1    Alanine Aminotransferase (ALT) ………………………………   67

3.9.2    Aspartate Amino Transferase (AST) Assay…………………  67

3.9.3    Determination of Alkaline Phosphatase (ALP)………………… 68

3.10     Statistical Analysis …………………………………………….    69

CHAPTER FOUR: RESULTS

4.1        Results…………………………………………………………………………….   70

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS 

5.1       Discussion………………………………………………………       86

5.2       Conclusion……………………………………………………      95

5.3       Recommendations  ………………………………………………        96

REFERENCES…………………………………………………………………………    97

APPENDIX………………………………………………………………………………   115

LIST OF TABLES

Table 3.1 Feeding of the Animals……………………………………………  61

Table 4.1 Proximate Composition of Jatropha curcas & Brillantasia nitens leaves Extracts……………………………………            70

Table 4.2 Phytochemical Composition of Jatropha curcas &Brillantasia nitens Leaves Extracts…………………………            70

Table 4.3 Antinutrient Contents of Jatropha curcas & Brillantasia nitens Leaves Extracts………        71  

Table 4.4: Mineral Composition of Jatropha curcas and Brillantasia nitens Leaves Extracts… …     71

Table 4.5: Vitamin Contents of Jatropha curcas and Brillantasia nitens Leaves Extracts………        72

Table 4.6: Mean Body Weight of Rats in Different Groups Fed Animal Feed Supplemented

with 100-300mg of Jatropha curcas and Brillantasia nitens………………………….     73

Table 4.7: Packed Cell Volume Level of Rats Fed Levels of Jatropha curcas Leaf Extracts…           74

Table 4.8: Packed Cell Volume Level of Rats Fed Levels of Brillantasia nitens Leaf Extracts…        74

Table 4.9: Hemoglobin Concentration of Rats Fed Levels of Jatropha curcas Leaf Extract……        75

Table 4.10: Hemoglobin Concentration of Rats Fed Levels of Brillantasia nitens Leaf Extracts…     75

Table 4.11: Red Blood Cell Count of Rats Fed Levels of Jatropha curcas Leaf Extract………          76

Table 4.12: Red Blood Cell Count of Rats Fed Levels of Brillantasia nitens Leaf Extract. ……         76

Table 4.13: White Blood Cell Count of Rats Fed Levels of Jatropha curcas Leaves Extract………     77

Table 4.14: White Blood Cell Count of Rats Fed Level of Brillantasia nitens Leaves Extract…..       77

Table 4.15: Mean Corpuscular Volume of Rats Fed Levels of Jatropha curcas Leaf Extract …         78

Table 4.16: Mean Corpuscular Volume of Rats Fed Levels of Brillantasia nitens Leaves Extract.      78

Table 4.17: Mean Corpuscular Hemoglobin of Rats Fed Levels of Jatropha curcas Leaf Extract..     79

Table 4.18: Mean Corpuscular Hemoglobin of the Rats Fed Levels of Brillantasia nitens Leaf Extract……………………………………………        79

Table 4.1: Mean Corpuscular Hemoglobin Concentration Value of Rats Fed Levels of Jatropha curcas Leaf Extracts…………………………………. 80

Table 4.20: Mean Corpuscular Hemoglobin Concentration of the Rats Fed Different Levels of Brillantasia nitens Leaf Extract…………………………    80

Table 4.21: Effect of Jatropha curcas Leaf Extract on Aspartate Amino Transferase Activity in Anaemia Induced Rats…………………………        81

Table 4.22: Effect of Leaf Extract of Brillantasia nitens on Aspartate Amino Transferase Activity in Anaemic Induce Rats………………………    81

Table 4.23: Effect of Leaf Extract of Jatropha curcas on Alanine Amino Transferase  Activity Levels in Anaemic Induced Rats.……………… 82

Table 4.24: Effect of Leaf Extracts of Brillantasia nitens on Alanine Amino Transferae Activity Levels in Anaemic Induced Rats. ……………… 82

Table 4.25: Effect of Leaf Extract of Jatropha curcas on Alkaline Phosphatase Activity Levels in Anaemia Induced Rats….      83

Table 4.26: Effect of Leaf Extract of Brillantasia nitens on Alkaline Phosphatase Activity Levels in Anaemia Induced Rats.,……………………………  83

LIST OF FIGURE

Fig 3.1: Processing the two vegetables used in the study……..        51

  LIST OF PLATES

 A1: Jatropha curcas leaves………………………………………………        114

A2:Brillantasia nitens leaves……………………………………           114

ABSTRACTS

The study investigated micronutrient contents and effects of Jatropha curcas and Brillantasia nitens leaves extracts in anaemia induced albino adultmale rats. Thirty five male albino adult rats weighing between 90-155gwere purchased from Department of Zoology and Environmental Sciences,University of Nigeria, Nsukka. The rats were housed in individual metabolic cages. The rats were weighed and allotted into seven (7) groups of five (5) rats. The difference in weight of rats in each group was not more than five grammes (5g). Groups 2, 3, 4, 5, 6 and 7 were injected with cyclophosphamide to induce anaemia in the rats. Group one served as a control group without anaemia and was fed animal feed and water only. Six groups of rats were fed animal feed, water,Jatropha curcas and Brillantasia nitens leaves extracts. Groups 2- 4 were fed 100mg, 200mg and 300mg/kg body weight respectivelyof Jatropha curca leaf extract. Groups 5- 7 were fed 100mg, 200mg and 300mg/kg body weightrespectively of Brillantasia nitens leaf extract. Chemical and biochemical analyses were determined using standard method. The data were analyzed using Statistical Package for Service Solution (version 21) (SPSS). Duncan’s Studentised New Multiple Range Test was used to separate and compare means at 5% probability. The two extracts had high and comparable moisture contents (97.69 and 97.99%). They had low protein, ash, fat and carbohydrate. Phytochemical compositions were: 1.06% for alkaloid, 0.38% for flavonoid and 0.63% for saponins for Jatropha curcas.  Brillantasia nitens had 1.55% for alkaloid, 2.13% for flavonoid and 0.18% for saponins. Brillantasia nitens had phytate 18.83 mg and oxalate 0.22%. Jatropha curcas had higher value for iron 0.74mgand that of Brillantasia nitens was 0.08mg. The zinc content of Jatropha curcas was 0.10mg and 0.12mg for Brillantasia nitens.Brillantasia nitens leaves extracts had higher values in all the vitamins analysized,  42.13mg for vitamin C, 629.00IU/L for beta carotene, 0.78 mg for vitamin E, 2.83 mg for vitamin B₆ and 672.00µg for vitamin B₉.The extracts tremendously increased the packed cell volume of all the rats. The highest increase was that of group 4 fed 300mg of Brillantasia nitens followed by 200mg of same leaf extract (70.32% and 58.78% respectively). There were slight increases in hemoglobin (Hb) concentration of rats fed Jatropha curcas. The rats fed diets 300mg of Brillantasia nitens had the highest increase in heamoglobin (70.71%). The extracts increased the red blood cell count of all the rats. The white blood cell increased in rats fed 200 and 300mg of Jatropha curcas (4.86% and 14.93% respectively). There was a decrease in white blood cell of all rats fed Brillantasia nitens leaf extract. All groups of rats had an increase in alanine amino transferase (ALT) activities and deceases in aspartate amino-transferase (AST) and alkaline phosphatase (ALP) activities. The haematinic potential of these extracts maybe due to the phytochemical component of the extracts that increased the haematological indices. The micronutrient constituents of the extracts appear to be the major possible active component accountable for the haematinic effect. Jatropha curcas and Brillantasia nitens leave extracts had high nutritive value and as such could be use in management of anaemia.

Keywords: Jatropha curcas,Brillantasia nitens, anaemia, micronutrient constituents, haematological

CHAPTER ONE

1.0   INTRODUCTION

1.1       Background to the Study

Vegetables are one of the most natural foods that contain different micronutrients and thousands of phytochemicals known for their health benefits. There is a wide variety of indigenous vegetables and fruits found in Africa, which are chief sources of micronutrients, antioxidants, and proteins (Odhav et al., 2007). Some of the indigenous vegetables and fruits are mainly used by inhabitants for medicinal purposes (Eifediyi et al., 2008). Leafy vegetables and herbs are relatively inexpensive.They are easy to prepare as rich sources of precursors of several nutrients, especially β-carotene.

Many indigenous plants (trees,shrubs, herbs, twigs and leafy vegetables) are consumed as food,spices or used for medicinal purposes in Nigeria (Nwaogu et al.,2007).Vegetables, especially tomatoes and leafy vegetables, fruits, dried beans and nuts serve as sources of iron (non-heme). In addition to the iron contained in vegetables, the high levels of vitamin C in many vegetables will increase the efficiency of dietary iron absorption(McKinley Health Center, 2010). Consumption of green leafy vegetables can reduce many micronutrient deficiency diseases. Green leafy vegetables are rich sources of carotenoids as well as iron, calcium, ascorbate, riboflavin, folate and appreciable amounts of other minerals.

Micronutrients are nutrients needed in minute specific quantities in the body. Most of them are not produced in the body. They are derived from food when consumed. Some of these micronutrients are vitamins A, B₁₂, iron, folate, iodine and zinc. Prolonged inadequate intake of foods rich in these micronutrients precipitates their deficiencies. One-third of the world’s population suffers from micronutrient deficiencies, due to inadequate dietary intake (Fielder and Macdonald, 2009). Micronutrient malnutrition is widespread in the industrialized nations and even more so in the developing regions of the world. This is due to endemic nature of malaria between 10 to 20% of the population presents less than 10 g/dl of haemoglobin (Diallo et al., 2008).

According to World Health Organization (2001), anaemia is defined as a hemoglobin concentration lower than the established cutoff. This cutoff figure ranges from 110 g/L for pregnant women and for children 6 months–60 months of age, to 130 g/L for men. Sex, age, and pregnancy status, other factors influence the cutoff values for hemoglobin concentration while others include altitude, race, and whether or not the individual smokes (WHO, 2001). Anaemia can be diagnosed by analyzing the hemoglobin concentration in blood or by measuring the proportion of red blood cells in whole blood (hematocrit). Nutritional anaemia is caused when there is an inadequate body store of a specific nutrient needed for hemoglobin synthesis. The most common nutrient deficiency is iron. Iron plays an important   role in the production of haemoglobin.

Iron deficiency is a condition in which the oxygen carrying capacity of the blood is reduced, indicating a sign of an underlying disease (Ochei and Kolhatkar, 2008). It occurs because of lack of the iron in the body. Iron deficiency is ranked at the top of three global “hidden hungers” (iron, iodine and vitamins A) with about one fifth of the world’s population suffering from iron deficiency anaemia (WHO, 2008a). Iron deficiency in its most severe form results in anaemia–Iron Deficiency Anaemia (IDA). The prevalence of anaemia has often been used as proxy of IDA (United Nations Children’s Fund (UNICEF),2004).

Forty percent of the world’s population (>2 billion individuals) suffer from anaemia (WHO, 1996), many are due to iron deficiency (UNICEF, 2004). Iron deficiency is estimated to be the most common cause of anaemia worldwide and is particularly prevalent in developing nations in Africa and Asia (Stoltfzfus et al, 2004). A review of national representative survey from 1993 to 2005 showed that 30% of non-pregnant women of childbearing age, 42% of pregnant women, and 47% of preschool children worldwide had anaemia (Kraemer andZimmermann, 2011). In developing countries, the prevalence of anaemia is 20% in children of school age (Echendu and Onimawo, 2003).

Anaemia constitutes a serious health problem in many tropical countries. This is because of the prevalence of malaria and other parasitic infection. In 31 of the 38 African countries that had data on iron deficiency, every secondchild under the age of 5 suffers from iron deficiency (FORTAF, 2000). In Nigeria, Anaemia Prevalence among Under-five Children in Imo State showed that 70.5% was anaemic and 48.1% were iron deficient(Onyemaobi and Onimawo, 2011).Anaemia precipitates decreased level of circulating haemoglobin, less than 13 g/dl in male and 12 g/dl in females (Okochi et al., 2003).The thrust of this investigation is to determine the micronutritent content and effect of extracts of Jatropha curcas and Brillantasia nitens in anaemia induced rats.       

1.2 Statement of the Problem          

Anaemia is an important public health problem in both developed and developing countries (Administrative Committeeon Coordination – Sub-Committee on Nutrition (ACC/SCN), 2004). It is a worldwide nutritional disorder that is highly prevalent in developing regions of the world and has been a major public-health problem affecting a greater percentage of the world’s population (Haidar, 2010). Most developing countries are battling with hunger, poverty and high rate of unemployment. These give rise to food insecurity in most of the households. Insufficient consumption of vegetables and fruits annually causes 2.7 million deaths worldwide, and is one of the top ten risk factors contributing to human mortality (Oladele,2011).Anaemia affects all age groups but is more concentrated in preschool aged children and women, making it a global health problem (McLeam, Co Swell, Egli, Wofdylay and De Benoist, 2009).

The prevalence of iron deficicency anaemia is at a greater rate in many developing countries including Nigeria especially among the low socio-economic class citizens who cannot afford the cost of sources from animal origin that are rich in iron. Deficiency of some micronutrient such as copper, vitamin A, vitamin B complex (folate and B₁₂) can increase the risk of anaemia. These deficiencies usually occur where diets are not diversified to include sufficient quantities of fruits, vegetables, dairy products, meats and fish which are best sources of micro-nutrients (Food and Agriculture Organization (FAO) 2002). The incidence of anaemia is likely to increase in future(Duff, 2008), therefore, the need arises to prevent it or seek for morecost-effective and better treatment strategies.

A good number of medicinal plants are traditionally employed to alleviate anaemia. Some vegetables have been successfully used locally to reverse cases of iron deficiency anaemia. These vegetable are lesser known and are more available during rainy season. Based on these observations, it is imperative that whole or extract of some common or lesser known vegetables be used as effective remedy to manage iron deficiency anaemia.It is therefore, very necessary to scientifically establish the micronutrient contents and effects of Jatropha curcas and Brillantasia nitens leaves extractsin anaemia induced rats.

1.3       Objective of the Study

            Broad Objective

The broad objective of this study was to determine the micronutrient content and effects of Jatropha curcas and Brillantasia nitens leaves extracts in anaemia induced adult male albino rats

Specific Objectives

The specific objectives of the study were to;

1. determine the proximate composition of Jatropha curcas and Brillantasia nitens leaveextract;
2. determine the micronutrient (iron, zinc, copper, beta carotene, vitamin C, E, B_6, and B₉) composition of Jatropha curcas and Brillantasia nitens leave extract;
3. determine the antinutrient (tannins, phytates and oxalate) contents of Jatropha curcas and Brillantasia nitens leave extract;
4. determine the phytochemicals (saponins, flavoniod and alkaloids) contents of Jatropha curcas and Brillantasia nitens leave extract;
5. evaluate the effects of the extracts on some haematological indices (packed cell volume (PCV), red blood cell (RBC), white blood cell (WBC), hemoglobin (Hb), mean corpuscular volume (MCV),mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)of the anaemia induced male rats;

• determine the liver function test (alanine amino transferase (ALT), aspartate aminotransferase (AST) andalkalinephosphatase (ALP)) of the anaemia induced male rats.