STUDIES ON THE PHYTOCHEMICAL AND NUTRITIONAL COMPOSITION OF AQUEOUS LEAF EXTRACT OF JUSTICIA CARNEA AND ITS EFFECT ON SOME BIOCHEMICAL PARAMETERS IN ANAEMIC RATS

ABSTRACT
Anaemia is a common blood disorder that affects people of all ages, though people at greater risk are the elderly, young women and infants. Different drugs are used but many depend mainly on plants and herbal products for its treatment. In this study, the aqueous leaf extract of Justicia carnea was used. The percentage yield of the extract was 18.43. The proximate constituents of the extract were 4.61 ± 0.14 % (moisture), 2.66 ± 0.31 % (fats), 9.26 ± 0.02 % (ash), 3.68 ± 0.23 % (crude fibre), 26.71 ± 0.02 % (protein) and 53.07 ± 0.07 % (carbohydrates). The qualitative and quantitative phytochemical screening showed that it contained: 9.19 ± 0.15 mg/g (phenol), 7.06 ± 0.05 mg/g (flavonoids), 5.91 ± 0.30 mg/g (reducing sugars), 5.77 ± 0.40 mg/g (alkaloids), 5.39 ± 0.49 mg/g (glycosides), 4.10 ± 0.09 mg/g (carbohydrate), 1.18 ± 0.01 mg/g (saponins) and 0.70 ± 0.00 mg/g (tannins). The anti-nutrientive constituents were 0.06 ± 0.21 mg/g (phytate), 0.06 ± 0.00 TIU/mg (trypsin inhibitor), 0.06 ± 0.04 mg/g (oxalate), 0.07 ± 0.00 mg/g (hydrogen cyanide) and 0.38 ± 0.02 HIU/mg (haemagglutinin); while the vitamin contents were 1.37 ± 0.40 mg/g (B1), 2.50 ± 0.50 mg/g (B2), 0.04 ± 0.00 mg/g (B6), 0.05 ± 0.00 mg/g (B12), 0.77 ± 0.03 mg/g (B9), 2.97 ± 0.05 µg/g (A), 36.69 ± 0.83 mg/g (C), 0.30 ± 0.00 mg/g (E) and some minerals include 8.61± 0.38 µg/g (iron), 0.03± 0.00 mg/g (copper), 1.96 ± 0.01 µg/g (zinc), 1.06 ± 0.01 % (magnesium) and 4.59 ± 0.00 % (calcium) were also present. The acute toxicity test of the aqueous leaf extract of Justicia carnea showed no death in the mice up to 5000 mg/kg body weight of the extract. To assess the effect of the aqueous leaf extract of Justicia carnea on selected haematological and biochemical parameters, animals used were grouped into seven of four rats each. Anaemia was induced using the method described by Stone, 1954 in five groups (2, 3, 4, 5 and 6) while group 1 (not treated) and group 7 (treated with 500 mg/kg body weight of the extract) were not induced with anaemia. A significant increase (p < 0.05) was observed in dose dependent manner in the PCV value of animals in groups 3, 4, 5, 6 and 7 (50 mg/kg b.w of astyfer, 100, 300, 500, 500 mg/kg b.w respectively of the extract), when compared to group 2 (induced with anaemia but not treated). Also, there was a significant increase (p < 0.05) in groups 1 and 7, significant decrease (p < 0.05) in group 2 when compared to group 3 (50 mg/kg Astyfer) on days four and eight post-treatment. There was a significant increase (p < 0.05) in the RBC count of animals in groups 1 and 7 when compared to the group 2. Similarly, there was a significant increase (p < 0.05) in groups 1 and 7 and non-significant decrease (p > 0.05) in groups 2, 4, 5 and 6 when compared to group 3. There was a significant increase (p < 0.05) in the Haemogloblin concentration of animals in groups 1, 5 and 7 when compared to group 2. There was a non-significant (p > 0.05) increase in group 5 when compared to group 3. There was an increase in the WBC count of animals in groups 1, 3, 4, 5 and 7 but was significant (p < 0.05) in groups 1 and 7 when compared to group 2. A significant increase (p < 0.05) was observed in group 1 when compared to group 3. The liver enzymes (alkaline phosphatas, alanine and alkaline transaminase) activities showed a non-significant increase (p > 0.05) when compared to group 1 on day fourteen post-treatment. Similarly, there was a non-significant increase (p > 0.05) in the urea and creatinine level of the treated anaemic rats when compared to group 1. From this study, it can be concluded that the leaves of Justicia carnea are rich in vitamins and minerals, possess no toxic effect and serves as an effective haematinic.

TABLE OF CONTENTS
PAGE
Title Page – – – – – – – – – i
Certification – – – – – – – – – ii
Dedication – – – – – – – – – iii
Acknowledgement – – – – – – – – – iv
Abstract – – – – – – – – – v
Table of contents – – – – – – – – – vi
List of figures – – – – – – – – – xii
List of tables – – – – – – – – – xiii
CHAPTER ONE: INTRODUCTION
1.1 Haematopoiesis – – – – – – – – 2
1.2 Erythropoiesis – – – – – – – – 2
1.2.1 Erythropoietin (EPO) – – – – – – – – 2
1.3 Anaemia – – – – – – – – 3
1.3.1 Aetiology of anaemia – – – – – – – – 4
1.3.2 Types and of anaemia – – – – – – – – 4
1.3.2.1 Iron deficiency anaemia – – – – – – – 4
1.3.2.2 Haemolytic anaemia – – – – – – – – 4
1.3.2.3 Sideroblastic anaemia – – – – – – – – 5
1.3.2.4 Pernicious anaemia – – – – – – – 5
1.3.2.5 Aplastic anaemia – – – – – – – 6
1.3.2.6 Megaloblastic anaemia – – – – – – – 6
1.3.2.7 Acute haemorrhagic anaemia – – – – – – – 6
1.4 Haematological Indices – – – – – – – 7
1.4.1 Red blood cells (erythrocytes) – – – – – – 7
1.4.2 Packed cell volume (PCV) – – – – – – 7
1.4.3 Haemoglobin – – – – – – – 7
1.4.4 White blood cells (WBCs) – – – – – – – 7
1.5 Justicia carnea leaves – – – – – – – 8
1.5.1 Medicinal properties of Justicia carnea – – – – – 8
1.5.2 Scientific classification of Justicia carnea – – – – – 9
1.6 Phytochemicals – – – – – – – 10
1.6.1 Alkaloids – – – – – – – – 10
1.6.2 Saponins – – – – – – – – 11
1.6.3 Phenols – – – – – – – – 11
1.6.4 Tannins – – – – – – – – 11
1.6.5 Flavonoids – – – – – – – – – 12
1.6.6 Terpenoids – – – – – – – – – 12
1.6.7 Glycosides – – – – – – – – – 12
1.7 Vitamins – – – – – – – 13
1.7.1 Vitamin A – – – – – – – – – 13
1.7.2 Vitamin C – – – – – – – – – 13
1.7.3 Vitamin E – – – – – – – – – 14
1.7.4 Vitamin B1 – – – – – – – – – 14
1.7.5 Vitamin B2 – – – – – – – – – 15
1.7.6 Vitamin B6 – – – – – – – – – 15
1.7.7 Vitamin B9 – – – – – – – – – 15
1.7.8 Vitamin B12 – – – – – – – – – 16
1.8 Minerals – – – – – – – – – 16
1.8.1 Calcium – – – – – – – – – 17
1.8.2 Magnesium – – – – – – – – – 17
1.8.3 Iron – – – – – – – – – – 17
1.8.4 Zinc – – – – – – – – – – 18
1.8.5 Copper – – – – – – – – – 18
1.9 The liver – – – – – – – – – 19
1.9.1 Serum enzymes marker involved in hepatic disorder – – – – 19
1.10 The Kidney – – – – – – – – – 19
1.11 Research objective – – – – – – – – 20

CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials – – – – – – – – – 21
2.1.1 Plant material – – – – – – – – 21
2.1.2 Animals – – – – – – – – 21
2.1.3 Equipment – – – – – – – – – 21
2.1.4 Chemicals and reagents – – – – – – – 22
2.2 Methods – – – – – – – – – 23
2.2.1 Preparation of plant material – – – – – – – 23
2.2.2 Extraction of plant material – – – – – – – 24
2.2.3 Preparation of reagents for phytochemical analysis – – – – 24
2.3 Proximate analysis of Justicia carnea – – – – 25 2.3.1 Determination of moisture content – – – – – – 26
2.3.2 Determination of fat content – – – – – – – 26
2.3.3 Determination of crude fibre content – – – – – – 26
2.3.4 Determination of ash content – – – – – – – 26
2.3.5 Determination of protein content – – – – – – 27
2.3.6 Determination of carbohydrate content – – – – – 27
2.4.1 Test for alkaloids – – – – – – – – 27
2.4.2 Test for flavonoids – – – – – – – – 28
2.4.3 Test for glycosides – – – – – – – 28
2.4.4 Test for proteins – – – – – 28
2.4.5 Test for carbohydrates – – – – – – – 28
2.4.6 Test for reducing sugars – – – – – 29
2.4.7 Test for saponin s – – – – – – – 29
2.4.8 Test for tannin s – – – – – – 29
2.4.9 Test for oil – – – – – – – 29
2.4.10 Test for resins – – – – – – – – 30
2.4.11 Test for terpenoids and steroid s – – – – – – 30
2.5 Quantitative phytochemical analysis of Justicia carnea – – 30
2.5.1 Determination of alkaloids – – – – – – 30
2.5.2 Determination of flavonoids – – – – – – – 31
2.5.3 Determination of reducing sugars – – – – – – 31
2.5.4 Determination of total phenol s – – – – – – 31
2.5.5 Determination of terpenoids – – – – – – – 31
2.6 Determination of anti-nutrient contents of Justicia carnea – – – 32
2.6.1 Tannins – – – – – – – – – 32
2.6.2 Phytate – – – – – – – – – 32
2.6.3 Cyanogenic glycosides – – – – – – – 32
2.6.4 Oxalates – – – – – – – – 33
2.7 Determination of vitamin content of Justicia carnea – – – – 33
2.7.1 Vitamin A – – – – – – – – – 33
2.7.2 Vitamin E – – – – – – – – 34
2.7.3 Vitamin C – – – – – – – – 34
2.7.4 Vitamin B1 – – – – – – – – – 34
2.7.5 Vitamin B2 – – – – – – – – – 34
2.7.6 Vitamin B12 – – – – – – – – – 35
2.8 Determination of some mineral components of Justicia carnea – – 35
2.8.1 Iron – – – – – – – – – 35
2.8.2 Zinc – – – – – – – – – 36
2.8.3 Calcium – – – – – – – – 36
2.8.4 Magnesium – – – – – – – – 36
2.9 Acute toxicity test of aqueous extract of Justicia carnea – – 36
2.10 Haematological study – – – – – – – 37
2.10.1 Experimental design for the haematological studies – – – – 37
2.11 Haematological parameters of rats treated with the aqueous extract of Justicia
carnea leaves. – – – – – – – – – 37
2.11.1 Determination of packed cell volume – – – – – – 37
2.11.2 Determination of haemoglobin (Hb) concentration – – – 38
2.11.3 Determination of red blood cells (RBCs) – – – 39
2.11.4 Determination of white blood cells (WBCs) – – – – 39
2.12 Liver function test of rats treated with aqueous extract of Justicia carnea leaves- 40
2.12.1 Assay for alanine aminotransferase (ALT) – – – 40
2.12.2 Assay for aspartate aminotransferase (AST) – – – 41
2.12.3 Assay for alkaline phosphatase (ALP) – – – – 41
2.13 Renal function test of rats fed aqueous extract of Justicia carnea leaves – 42
2.13.1 Determination of serum urea – – – – – – 42
2.13.2 Determination of serum creatinine – – – – – 43
2.14 Statistical Analysis – – – – – – – 43
CHAPTER THREE: RESULTS
3.1 Percentage yield of aqueous leaf extract of Justicia carnea 44
3.2 Qualitative phytochemical screening of Justicia carnea – – – 44
3.3 Quantitative phytochemical constituent of aqueous leaf extract of Justicia carnea -45
3.4 Proximate constituent of aqueous leaf extract of Justicia carnea 46
3.5 Antinutrient composition of aqueous leaf extract of Justicia carnea – – 47
3.6 Vitamin Contents of the aqueous Leaf Extract of Justicia carnea.48
3.7 Mineral contents of the aqueous leaf extract of Justicia carnea 49
3.8 Acute Toxicity (LD50) Test of Aqueous Leaf Extract of Justicia carnea – 50
3.9 Effect of aqueous leaf extract of Justicia carnea on the percentage packed cell volume (PCV) of rats before and after induction of anaemia – – 51
3.10 Effect of aqueous leaf extract of Justicia carnea on the red blood cell (RBC) count of rats before and after induction of anaemia – – – 53
3.11 Effect of aqueous leaf extract of Justicia carnea on the haemoglobin concentration (Hb Conc.) of rats before and after induction of anaemia – – 55
3.12 Effect of aqueous leaf extract of J. carnea on the white blood cell (WBC) count of rats before and after induction of anaemia – – – – 57
3.13 Effect of aqueous leaf extract of J. carnea on aspartate transaminase (AST) of rats – – – – – – – – – – 59
3.14 Effect of aqueous leaf extract of J. carnea on alanine transaminase (ALT) of rats 61
3.15 Effect of aqueous leaf extract of J. carnea on alkaline phosphatase (ALP) of rats 63
3.16 Effect of aqueous leaf extract of J. carnea on urea concentration of rats 65
3.17 Effect of aqueous leaf extract of J. carnea on creatinine concentration of rats 67

CHAPTER FOUR: DISCUSSION
4.1 Discussion – – – – – – – – – 69
4.2 Conclusion – – – – – – – – – 75
4.3 Suggestions for further studies – – – – – – – 75
REFERENCES – – – – – – – – 76
APPENDICIES – – – – – – – – 96

LIST OF FIGURES
Figure 1: Justicia carnea (J. carnea) – – – – – – 9
Figure 2. Effect of the aqueous leaf extract of J. carnea on the packed cell volume (PCV) of rats before and after induction of anaemia – – – 52
Figure 3. Effect of the aqueous leaves extract of J. carnea on the red blood cell (RBC) count of rats before and after induction of anaemia – – – 54
Figure 4. Effect of the aqueous leaf extract of J. carnea on the haemoglobin concentration (Hb Conc.) of rats before and after induction of anaemia – – – – – 56
Figure 5. Effect of the aqueous leaf extract of J. carnea on the white blood cell (WBC) count of rats before and after induction of anaemia – – – 58
Figure 6. Effect of aqueous leaf extract of J. carnea on aspartate transaminase (AST) activity of rats – – – – – – – – – – – – 60
Figure 7. Effect of aqueous leaf extract of J. carnea on alanine transaminase (ALT) activity of rats – – – – – – – – – – – 62
Figure 8. Effect of aqueous leaf extract of J. carnea on the alkaline phosphatase (ALP) activity of rats – – – – – – – – – – 64
Figure 9. Effect of the aqueous leaf extracts of J. carnea on the urea concentration of rats – 66
Figure 10. Effect of the aqueous leaf extract of J. carnea on creatinine concentration of rats 68

LIST OF TABLES
Table 2: Result of phytochemical screening of aqueous leaf extract of J. carnea 44
Table 3: Quantitative phytochemical constituents of aqueous leaf extract of J. carnea 45
Table 4: Proximate constituents of aqueous leaf extract of J. carnea 46
Table 5: Anti-nutrient composition of the aqueous leaf extract of J. carnea 47
Table 6: Vitamin constituents of aqueous leaf extract of J. carnea 48
Table 7: Mineral constituents of aqueous leaf extract of J. carnea 49
Table 8: Phase I and II of the acute toxicity (LD50) test of J. carnea leaf extract 50

CHAPTER ONE
INTRODUCTION
Blood is a bodily fluid that delivers necessary substances such as nutrients and oxygen to the cells and transport metabolic waste products away from those same cells. There are three major categories, each of which has specific functions (Ballard, 1997). The white blood cell (WBCs), or leukocytes, engage in defense of the body against foreign microorganisms or toxic substances as well as mediate immune response. The WBCs are sub-divided further into three groups; granulocytes, monocytes and macrophages, and lymphocytes (T-cells and B-cells). The platelets help maintain the integrity of the blood vessels by stimulating blood clotting (i.e., coagulation) after an injury. Red blood cells (RBCs), also called erythrocytes, transport oxygen from the lungs to all the cells in the body and carry carbon dioxide from the cells back to the lungs (Ballard, 1997).
Anaemia is a widespread public health problem associated with an increased risk of morbidity and mortality, especially in pregnant women and young children (WHO, 2002). Among the numerous factors, both nutritional (such as vitamin and mineral deficiencies) and non-nutritional (such as infection and haemoglobinopathies), that contribute to the onset of anaemia, iron deficiency and malaria play an important role. Others include; inherited genetic defects, medication-related side effects, and chronic disease (WHO, 2002).
Man has been using herbs and plant products in combating diseases since time immemorial. The traditional system of medicine has been such that larger percentages of the populations in Africa and Asia depend on indigenous system for relief of symptoms of various diseases. Regular consumption of plant foods are associated with numerous health benefits rooted in their various physiological effects as a result of their phytochemical and nutritional constituents (Hunter and Fletcher, 2002). Green leafy vegetables are particularly important in promoting health because of their rich content of nutrients (Gupta and Prakash, 2009). A good number of medicinal plants are traditionally employed in alleviating anaemia. Some of these plants include; Telfeira occidentalis, Combretum dolichopetalum, Psorospermum ferbrifugum, Jatropha curcas, Flacourtia flavenscens and Brillantasia nitens (Alada, 2000; Dina et al., 2006) and Justicia carnea. The leaves of Justicia carnea are commonly used as haematinic and claimed to be very effective. This has led to the biochemiocal investigation the plant.
1.1 Haematopoiesis