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.

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

Haematopoiesis is the process by which all lineages of blood cells are generated in a hierarchical and stepwise manner from immature cells present in the bone marrow and subsequently released into circulating blood and peripheral organs for further maturation steps and/or effector function (Orikin and Zon, 2008). The production of all types of blood cells begins with undifferentiated precursor cells called pluripotent stem cells (Ballard, 1997). Though the different blood cells have distinct structures and functions, they are all produced at the same site, the bone marrow.

1.2 Erythropoiesis

Erythropoiesis is the process whereby erythroid precursor cells proliferate and differentiate into red blood cells (Smith, 1995). During the middle trimester of gestation, the liver is the main organ for production of red blood cells, but reasonable numbers are also produced in the spleen and lymph nodes. Then, during the last month of gestation and after birth, red blood cells are produced exclusively in the  bone  marrow. Even in these  bones,  the  marrow becomes less productive as age increases (Alayash, 2004).

RBC is both structurally and metabolically the simplest cell in the body – the end product of the maturation of bone-marrow reticulocytes (Smith, 1995). During its maturation, the RBC loses all its  subcellular organelles. Without  nuclei,  it  lacks  the  ability  to  synthesize DNA or  RNA. Without ribosomes or an endoplasmic reticulum, it cannot synthesize or secrete protein (Smith,

1995). Because it cannot oxidize fats, a process requiring mitochondrial activity, the RBC relies exclusively on blood glucose which is entirely anaerobic. RBC functions in delivering oxygen from  the  lungs  to  the  tissues  and  carbon  dioxide  from  the  tissues  to  the  lungs;  this  is accomplished by a tetramer protein known as haemoglobin (Hb) (Alayash, 2004).

1.2.1 Erythropoietin (EPO)

EPO is a 30.4-kD glycoprotein with four carbohydrate residues (Fried, 2009). It is a primary regulator  of  RBC  formation  (Elliot,  2008).  The  primary  site  of  EPO  production  in  adult mammals is the kidney although is also made at lower levels in other tissues such as liver and brain (Elliot, 2008). It is primarily regulated by oxygen supply to a sensor in the kidney relative to its oxygen requirements. Conditions that result in a decrease in oxygen delivery to the renal

sensor will stimulate EPO production and this includes residence at high altitude, pulmonary disease that affects the diffusing capacity of the lungs and results in decreased uptake of oxygen, admixture of arterial and venous blood through a right-to-left shunt, decreased renal blood flow, etc. (Fried, 2009). Conditions that increase oxygen delivery will decrease production of EPO (Fried,  2009).  In  conditions  that  decrease  the  oxygen  needs,  such  as  hypothyroidism and hypopituitarism,  a  normal  rate  of  oxygen  delivery  represents  plethora  and  causes  EPO production and the serum EPO titer to decline (Elliot, 2008).

1.3 Anaemia

Anaemia is one of the most common haematological defects affecting people of all ages, is a condition in which the number of red blood cells (RBCs) and also the amount of circulating haemoglobin is insufficient to meet the body’s physiological need (McGrath, 1993; Grewal,

2010). Anaemia is not considered a disease itself; rather, it reflects an abnormality in the number, structure, or function of RBCs (Rosdahl and Kowalski, 2008). Reduction of haemoglobin is usually accompanied by a fall in red cell count and packed cell volume (PCV) but these amount may be normal in some patients with subnormal haemoglobin level (Hoffbrand et al., 2006). Non-nutritional anaemia (such as sickle-cell anaemia and thalassaemia, which are induced by genetic disorders) is suffered most  by people in Sub-Saharan Africa,  but  these are  few  in comparison to the number of people with nutritional anemia. Some vitamins like vitamin B12, folic acid and riboflavin influence the formation of haemoglobin (Hoffbrand et al., 2006).

The  incidence  of anaemia  is  more  common  in  underdeveloped countries  because  of poor nutrition and the presence of parasites that extract blood from the intestines. The severity of anaemia depends on factors such as its speed of onset i.e. whether it is chronic, the person’s overall health and nutritional status. The more rapidly anaemia develops, the more likely it is to be serious (Rosdahl and Kowalski, 2008).

1.3.1 Aetiology of Anaemia

Anaemia  may  be  due  to  a  single  factor  such  as  a  nutritional deficiency  or  multifactorial

(McEvoy, 2013). It results from one or more of the following events: loss of red blood cells

(RBCs), reduction in the production of RBCs, increased destruction of RBCs, and shorter life span of RBCs (McEvoy, 2013). In men and post-menopausal women, anaemia is usually due to gastrointestinal blood loss associated with ulcers, use of aspirin or non-steroidal anti- inflammatory medications (NSAIDS), and parasite infestations. Bleeding episodes, vitamin deficiencies (e.g., of cobalamin and folic acid), hyper-splenism, autoimmune haemolysis, renal dysfunction, and radio-chemotherapeutic intervention themselves can also aggravate anaemia (Weiss and Goodnough, 2005). Multiple factors include: nutritional deficiencies, suppression of RBC production by medications, inflammatory cytokines (inflammatory anaemia or anaemia of chronic disease), phlebotomy, and chronic bleeding (McEvoy, 2013).

1.3.2 Types of Anaemia

1.3.2.1 Iron Deficiency Anaemia

Iron deficiency is the most common nutritional disorder, affecting more than one-third of the general  population  (Andrews,  1999;  Zimmermann  and  Hurell,  2007;  Clark,  2009).  Iron deficiency anaemia is the most prevalent anaemia in all age groups in the world. It results from either an inadequate absorption or an excessive loss of iron. This condition occurs most often in women, young children, and older adults. Primary causes include trauma, excessive menstrual, bleeding from the gastrointestinal tract, pregnancy, or a diet that lacks iron and poor absorption of iron by the body (Zimmermann and Hurell, 2007). It is also caused by lead poisoning in children (UNICEF, 2001). Deficiency caused by faulty eating habit is especially prevalent in the adolescent and elderly population (Clark, 2009).

1.3.2.2 Haemolytic Anaemia

Haemolytic  anaemia  results  from an  increase  in  the  rate  of red  cell  destruction.  Red  cell destruction  usually  occurs  after  a  mean  lifespan  of  120  days.  Because  of  erythropoietic hyperplasia and anatomical extension of bone marrow, red cell destruction may be increased several-fold before the patient becomes anaemic (Delaunay, 2003). The individual will have an increase of immature RBCs (reticulocytes). The cause of haemolytic anaemia is related to defects of the RBC cell membrane, inherited enzyme defects, certain drugs and toxins, antibodies, or physical trauma (Delaunay, 2003). Haemolytic anaemia may not be noticed until the red cell

lifespan is less than 30 days. Treatment of this type of anaemia relates to diagnosis and to the causative factors.

1.3.2.3 Sideroblastic Anaemia

Sideroblastic anaemia is a form of anaemia in which the bone marrow produces ringed sideroblasts rather than healthy red blood cells (Caudill et al., 2008). It may be caused by either genetic disorder or indirectly as part of myelodysplastic syndrome. Sometimes, however, the iron is not incorporated properly into the haemoglobin molecules. Sideroblast are atypical abnormal nucleated erythroblasts with granules of iron accumulated in perinuclear mitochondria and they are seen as aspirates of bone marrow (Caudill et al., 2008). Iron is converted into a storage form called ferritin, this accumulated ferritin often forms granules that encircle the cell’s nucleus. These ferritin- containing cells are called ringed sideroblasts and cannot mature (Ballard, 1997).

1.3.2.4 Pernicious Anaemia

Pernicious anaemia is the end stage of an autoimmune disorder in which parietal cell auto- antibodies against H+, K+-adenosine triphosphatase cause loss of gastric parietal cells. The loss of parietal cells reduces and then completely prevents production of intrinsic factor. In addition, blocking autoantibodies can bind to the B12  binding site for intrinsic factor and prevent the formation of the B12-intrinsic factor complex. Deficiency of intrinsic factor gradually results in B12  deficiency (Clarke et al., 2004). Approximately 20 percent of relatives of patients with pernicious anaemia also have pernicious anaemia (Toh et al., 1997). Pernicious anaemia carries an excess risk of gastric carcinoma (1 to 3 percent) and of gastric carcinoid tumors (Hsing et al.,

1993). Juvenile pernicious anaemia, a rare congenital disorder in which the stomach secretes abnormal intrinsic factor, generally affects children younger than 10 years (Clarke et al., 2004).

1.3.2.5 Aplastic Anaemia

Aplastic anaemia or bone marrow depression, describes a condition in which the bone marrow is underdeveloped  or  has  failed,  resulting  in  a   decrease  in  RBCs,  WBCs  and  platelets

(pancytopenia) (Young, 2000). This condition may occur at any age and develop very slowly or could be rapid and very severe. Causes include excessive radiation, toxicity to various drugs, tumors, insecticides, chemical, and environmental toxins (Marsh, 2005). It may develop as a complication of viral infections such as hepatitis, HIV/AIDS, and mononucleosis. In many cases, the cause is unknown (idiopathic aplastic anaemia). Bone marrow and stem cell transplantation is the most successful therapy (Young, 2000).

1.3.2.6 Megaloblastic Anaemia

Megaloblastic  anaemia  results  from  inhibition  of  DNA  synthesis  during  red  blood  cell production. When DNA synthesis is impaired, the cell cycle cannot progress from G2 growth stage to the mitosis stage, this leads to no division which presents as macrocytosis (Ballard,

1997). The defect in red cell DNA synthesis is most often due to hypovitaminosis, specifically B12  and or folic acid. Under conditions of folic acid deficiency, precursor cells cannot divide properly and large immature and nonfunctional cells (i.e., megaloblasts) accumulate in the bone marrow as well as in the bloodstream. This impaired haematopoiesis affects mainly RBCs’, but also WBCs’ and platelets. The most common cause of this deficiency is a diet poor in folic acid. (Ballard, 1997).

1.3.2.7 Acute Haemorrhagic Anaemia

Acute haemorrhagic anaemia develops after a rapid and often sudden blood loss. Causes of such blood loss include trauma that leads to blood vessel rupture, aneurysm, or artery erosion caused by a cancerous lesion or ulcer (Erber and Perry, 2006). Severity and prognosis depend on the total blood volume loss. A total blood volume loss of 20 % is considered a marked insufficiency. A loss of 30 % will cause failure in the circulatory system, coma, and shock. A loss that reaches

40 % can be fatal. Immediate treatment is volume replacement, often with administration of intravenous fluids, such as saline, albumin, or plasma, and transfusions with fresh whole blood (Nascimento et al., 2010).

1.4 Haematological Indices

Haematological indices are the parameters that are used in the assessment of the status of blood in the body. These parameters include red blood cells, packed cell volume, haemoglobin count,

total white blood cell, differential white blood cell and erythrocyte sedimentation rate (Jelkmann,

2004).

1.4.1 Red Blood Cells (erythrocytes)

Red blood cells are also known as erythrocytes. The cells are flexible biconcave disc that are involved in the transport of oxygen. The red blood cells carry haemoglobin into close contact with the tissues and for successful gaseous exchange, the red cell, 8µm in diameter, must be able to pass repeatedly through the microcirculation whose minimum diameter is 3.5µm, to maintain haemoglobin in a reduced (ferrous) state (Hoffbrand et al., 2006).

1.4.2 Packed Cell Volume (PCV)

Packed cell volume is a percentage of the known volume of whole blood occupied by packed blood cells  when the  blood is  centrifuged at  a constant speed and period of time. It  is  a reasonable index of the red cell population. It is normally 45 % for men and 40 % for women (Jelkmann, 2004).

1.4.3 Haemoglobin

Haemoglobin is a specialized protein that is contained in red cells of the blood. It is the actual protein in RBC that transports oxygen to tissues and carbon dioxide to the lungs. Each red cell contains approximately 640 million haemoglobin molecules (Hoffbrand et al., 2006). Haemoglobin consists of four polypeptide chains, α2β2, with each containing individual haem group. Haem synthesis occurs largely in the  mitochondria through a  series of biochemical reactions (Jelkmann, 2004).

1.4.4 White Blood Cells (WBCs)

White blood cells (leukocytes) are the cells of the immune system. They defend the body against pathogens, infections and foreign materials (Stock and Hoffman, 2000). White blood cells are of

two broad groups: the phagocytes and the immunocytes. The phagocytes include three cell types

– neutrophils (polymorphs), eosinophils and basophils and also the monocytes. The immunocytes include the lymphocytes, their precursor cell and plasma cells (Stock and Hoffman, 2000).

1.5 Justicia carnea

The genus Justicia, named after an 18th century Scottish botanist James Justice (1698-1763) belongs to the family of Acanthaceae consisting about 600 species of herbs, shrubs and tender perennial native to the tropics and subtropics (Correa, 2012).  Some common names for these tender perennials from Brazil and South America include: Brazilian plume, flamingo flower, Jacobinia, pine-bur begonia, pink jacobinia, pink tongues, king’s crown and cardinal’s guard. They come in varying heights, ranging from around 1.8m to 1m (Wasshausen and Wood, 2012). Their shapes vary from narrow upright shrubs to more rounded shrubs. The flowers are tubes, flared at the mouth and curving outward from the center of the spike on which the flowers are arranged. Justicia is easy to grow and propagate from stem cuttings by pushing the stems 1 to 2 inches into potting soil and kept moist until new leaves appear (Mabberley, 1997).

1.5.1 Medicinal Properties of Justicia carnea

Justicia carnea is a plant used for many purposes, it majorly serves as an ornamental plant (Parker and Pearson, 2012). Several species of Justicia are widely used in folk medicine for the treatment of inflammation, respiratory, and gastrointestinal disorder (Correa, 2012). Most of the medicinal properties such as antimicrobial, antioxidant, hypocholesterolemic and anti-cancerous are associated with its bioactive compounds mainly phenols and flavonoids (Bajpai et al., 2005). It has also been reported to be rich in both macronutrients and trace elements of which calcium and iron are in high quantity (Faiza et al., 2013). Justicia carnea is used as blood tonic in many parts of the country including Nigeria and this has led to more studies on the plant.

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