IN-VITRO ANTISICKLING EFFECT OF AQUEOUS EXTRACT OF SPHENOSTYLIS STENOCARPA SEED (AFRICAN YAM BEANS)

ABSTRACT

Sickle cell disease (SCD) is a major problem of the developing world. The search for anti- sickling agent is of particular interest since plant bioactive agents are said to be of medicinal significance. This research investigated aqueous extract of Sphenostylis stenocarpa seed for possible in-vitro anti-sickling properties. Induction of sickling and polymerization of HbSS was done by addition of 3.4ml of metabisulphite (Na2S2O2) solution. Further analyzes carried out on the extract include: phytochemical analyses, proximate analyses, mineral composition, vitamin composition, amino acid profile and antioxidant studies.The phytochemical screening of seed extract indicated the presence of steroids, saponin, hydrogen cyanide, terpenoid, and glycoside in relative amount while alkaloids, and terpenoids, at moderate level. Flavonoids, soluble carbohydrate,  tannin,  alkaloid  and  phenolics  were  present  in  high  amount.  The  proximate analysis  conducted  on  the  Sphenostylis  stenocarpa  seed  showed  the  nutrient  composition reported in descending order of magnitude thus; carbohydrate (63.24 ± 0.04%) > protein (20.41 ± 0.00%) > moisture (7.32 ± 0.03%) >fibre (3.84 ± 0.00%) >ash (3.78 ± 0.00) >fat (1.44 ±

0.00%). The vitamin content of the seed extract showed that it contained relative amount of vitamin  A,  C,  B1,  B2   and  B9.  The  result  revealed  the  presence  of  high  content  of  some antisickling  amino  acid  such  as  Phenylalanine  (4.75  ±  0.14%),  Lysine  (5.86  ±  0.05%), Asparagine (3.86 ± 0.02%), Serine (1.72 ± 0.07%), and Arginine (1.20 ± 0.05%) and a powerful antioxidant  potential.The effects  of the extract  on  membrane stabilization  and  inhibition of hemolysis of sickled RBCs showed that there was significant (p<0.05) increase among group 5,

6, 7, and 8 based on various concentrations of extract. The standard drug also showed that there was a significant (p <0.05) increase among the group 1, 2, 3, and 4. However, there exist also significant difference (p <0.05) between extract and standard (indomethacin) but exception of group 1 and 6 that showed non-significant (p >0.05) increase. The extract showed percentage inhibition of polymerization of sickle RBCs when compared with standard (P-hydroxybenzoic acid) based on increasing time interval. Group 1 percentage inhibition at 90th  second, 120th second and  180th  second showed no significant (p >0.05) difference but were found to be significantly  (p  <0.05)  higher  than  percentage  inhibition  of  30th   second  and  60th   second. However, group2 and 3 at 30th second were non-significant (p >0.05) when compared but were found to be significantly (p <0.05) lower than group 1 (positive control). Group 2 and 3 at 60th second revealed no significant (p >0.05) difference between the groups but were found to be significantly (p <0.05) lower than group 1. At 90th second, polymerization of sickling was found to reduce significantly (p <0.05) between group 1, 2 and 3. At 120th and 180th second, there was also significant (p <0.05) decrease between group 1, 2 and 3. Effect of the extract on sickling showed that there was significant (p <0.05) decrease of percentage sickling in group 3, 4 and 5 with increasing concentration of extract respectively. Sickling decreased from 36.84±0.30% to 7.89±0.31% based  on increasing  concentrations  of extract.  Group  6, 7, and 8  also showed significant (p <0.05) decrease with increase in concentrations of standard (P- hydroxybenzoicacid), from 36.84±0.30% to 9.47±0.30%. However, there was non-significant difference between group 3 (extract) and group 7 (standard). This legume could, therefore has immense nutritional and therapeutic importance in the management of sickle cell disease and other related diseases.

CHAPTER ONE

INTRODUCTION

Sickle cell disease (SCD) is a group of blood disorders typically inherited from a person’s parents. The most common type is known as sickle cell anemia (SCA).SCA is most prevalent with clinical manifestations attributed to a point mutation in the amino acid globin chain of hemoglobin A by the substitution of a hydrophilic glutamic acid residue for a hydrophobic valine residue at the sixth position of the β-chain of hemoglobin molecule (Ojiako et al., 2012). This substitution causes a drastic reduction in the solubility of sickle cell hemoglobin when deoxygenated. It results in an abnormality in the oxygen-carrying protein hemoglobin found in red blood cells. This leads to a rigid, sickle-like shape under certain circumstances.Problems in sickle cell disease typically begin around 5 to 6 months of age. Patients with acute manifestations may have prolonged and repeated hospitalization leading topoor quality of life and profound psychological impact. Multiple organ systems may be involvedleading to splenic infarction, leg ulcers,  pulmonary  hypertension,  strokes,  retinopathy,  attacks  of  pain  (“sickle  cell  crisis”), anemia, swelling in the hands and feet, bacterial infections, and vascular necrosis (Raghupathy and Bilett, 2009). The average life expectancy in the developed world is 40 to 60 years.

Drapanocytosis or sickle cell anemia is among the commonest genetic disorder in Sub- saharanAfrica and  Middle East responsible for great mortality. According to Mpiana  et al, (2009), thisdisorder was initially thought to exist in tropical and Mediterranean regions. Sickle cell disease(SCD) was first discovered by a Chicago physician, Dr James B. Herrick in 1904 when heexamined a 20year old black student from West Indies (Hammerschmidt, 2002). Normal redblood cells move through small vessels in the body to deliver oxygen and food nutrient. Sicklered blood cells, however, tend to obstruct the blood flow causing poor blood microcirculation(Kuyper et.al., 1994). The red cell membrane of sickle hemoglobin (HbSS) are osmotically andmechanically more fragile than those of hemoglobin AA(HbAA), hence sickle red blood cells are easily destroyed and removed from circulation in the spleen thus causing anemia andsubsequent splenomegally. In these conditions, the HbSS molecules polymerize to form long crystalline intracellular mass of fibers which are responsible for the deformation of the biconcave disc shaped erythrocyte into a sickle shape.

Africansremain the most affected by this disorder with the highest prevalence in West and Central Africa(Mpiana, et.al, 2009). In Nigeria, more than 3 % of its population is affected (Ibrahim et al.,2007) and about 80 % of children suffering from drapanocytosis that do not receive  regularmedical  care,  die  before  the  age  of  five  (Mpiana  et.al.,  2007).Most  of  the proposed therapies for sickle cell anemia (SCA) appear to be unsatisfactory. Bone marrow transplantation is expensive for African rural populations, fetal hemoglobin synthesis stimulants such  as  hydroxyurea  are  toxic  and  repeated  transfusions  constitute  high  risk  of  human immunodeficiency virus (HIV) infection (Akinsule et al., 2005). The cost of managing SCD is very high compared to the normal health care cost of non-sickle cell patient. The people living in the rural communities are mostly peasant farmers who may not afford the high cost of orthodox treatment for SCD. Due to the debilitating effects and the cost of managing SCD, researches are ongoing to determine the efficacy of the use of medicinal plants to tackle the multiple challenges of SCD (Okpuzoret al., 2008). Recent discoveries of antisickling phytoremedies that are cheaper and less toxic alternative therapies for SCD include: Piper guinesis, Pterocarpa osun, Eugenia Caryophlalaand  Sorghum  bicolour  extracts  (Wambebe  et  al.,  2001).  Different  species  of legumes abound in the tropical Africa, which are very rich sources of proteins and amino acids. Some  of  these  amino  acids  such  as  phenylalanine,  lysine,  arginine  and  glutamine  have antisickling properties (Ekeke et al., 2000; Nwaoguikpe and Uwakwe, 2005; Amehet al., 2012). This has led to the formation of an antisickling preparation ‘ciklavit’ in combination with other food extracts used in Nigeria and other West African countries for the management of SCD (Ekeke et al., 2000).

1.1 THE RED BLOOD CELL

A type of blood cell, also called erythrocyte that is made in the bone marrow and found in the blood. Red blood cells contain a protein called hemoglobin, which carries oxygen from the lungs to all parts of the body and also constitute its bright red colour.Hemoglobin contains the element Iron, making it an excellent vehicle for transporting oxygen and carbon dioxide. As blood passes through the lungs, oxygen molecules attach to the hemoglobin. As the blood passes through the body’s tissue, the hemoglobin releases the oxygen to the cells. The empty hemoglobin molecules then bond with the tissue’s carbon dioxide or other waste gases, transporting it away.

1.1.1 SHAPE AND SIZE OF ERYTHROCYTE

Mature RBCs are small, disc-shaped cells that measure 7-8 micrometers (µm) in diameter. As they mature, RBCs extrude their nucleus, which causes the centre of the cell to collapse.The resulting  biconcave  shape  gives  RBCs  more  surface  area  than  spherical  cells  of  the  same size.This modification allows O2 and CO2 to move more quickly through the plasma membrane (Uzoigwe C, 2006). The raised edges that are created when the cell collapses measure about 2-3 micrometers in thickness.Adult humans have roughly 2-3×10^13 (20-30 trillion) red blood cells at any given time, comprising approximately one quarter of the total human body cell number. Women have about 4-5 million erythrocyte per cubic millimeter of blood and men about 5-6 million per cubic millimeter, people living at high altitude with low oxygen tension will have more (Hillman et al., 2005).

In Infants, the count is 6 to 7 million per cubic millimeter and in fetus 7.8 million per cubic millimeter. In the first ten days of postnatal life, large number of RBCs is destroyed.

Fig.1. Shape and size of erythrocyte

Source: Uzoigwe C, 2006.

1.1.2 ERYTHROPOIESIS

Erythropoiesis  is  the  process  by  which  red  blood  cells  (erythrocytes)  are  produced.  It  is stimulated by decreased O2  in circulation, which is detected by the kidneys, which then secrete the hormone erythropoietin (Sherwood et al., 2005). This hormone stimulates proliferation and differentiation of red cell precursors, which activates increased erythropoiesis in the hemopoietic tissues, ultimately producing red blood cells. In postnatalbirds and mammals (including humans), this usually occurs within the red bone marrow. In the early fetus, erythropoiesis takes place in the mesodermal cells of the yolk sac. By the third or fourth month, erythropoiesis moves to the spleen and liver (Palis and Segal, 1998). After seven months, erythropoiesis occurs in the bone marrow. Increased level of physical activity can cause an increase in erythropoiesis (Le et al.,

2010). However, in humans with certain diseases and in some animals, erythropoiesis also occurs

outside   the   bone   marrow,   within   the   spleen   or   liver.   This   is   termed   extramedullary erythropoiesis.

The bone marrow, essentially all the bones produces RBCs until a person is around five years old. The tibia and femur cease to be important sites of hematopoiesis by about age 25; the vertebrae, sternum, pelvis and ribs, and cranial bones continue to produce red blood cells throughout life.

1.1.2.1 ERYTHROCYTE DIFFERENTIATION

In the process of red blood cell maturation, a cell undergoes a series of differentiations. The following stages of development all occur within the bone marrow:

1.    Hemocytoblast, a multipotenthematopoieticstem cell

2.    Common Myeloid Progenitor, a multipotent stem cell

3.    Unipotent Stem Cell

4.    Pronormoblast, also commonly called Proerythroblast or Rubriblast.

5.    Basophilic Normoblast/Early Normoblast, also commonly called Erythroblast

6.    Polychromatophilic Normoblast/Intermediate Normoblast

7.    Reticulocyte

The cell is released from the bone marrow after Stage 7, and so of circulating red blood cells there are 1% reticulocytes. After one to two days, these ultimately become “erythrocytes” or mature red blood cells. These stages correspond to specific appearances of the cell when stained with Wright’s stain and examined by light microscopy, but correspond to other biochemical changes.  In the process of maturation, a Basophilic Pronormoblast is converted from a cell with a large nucleus and a volume of 900fL to an enucleated disc with a volume of 95fL. At the reticulocyte stage, the cell has extruded its nucleus, but is still capable of producing hemoglobin. Essential for the maturation of RBC’S are Vitamin B12 (cobalamin) and Vitamin B9 (Folic acid). Lack  of  either  of  these  causes  maturation  failure  in  the  process  of  erythropoiesis,  which manifests clinically as reticulocytopenia, an abnormally low amount

Source:( Paliset al.,1998).

1.1.2.2 REGULATION OF ERYTHROPOIESIS

A feedback loop involving erythropoietin helps regulate the process of erythropoiesis so that, in non-disease states, the production of red blood cells is equal to the destruction of red blood cells and the red blood cell number is sufficient to sustain adequate tissue oxygen levels but not so high as to cause sludging, thrombosis, or stroke. Erythropoietin is produced in the kidney and liver in response to low oxygen levels. In addition, erythropoietin is bound by circulating red blood cells; low circulating numbers lead to a relatively high level of unbound erythropoietin, which stimulates production in the bone marrow(Lankhorst and Wish, 2010).

Recent studies have also shown that the peptide hormone hepcidin may play a role in the regulation of hemoglobin production, and thus affect erythropoiesis. The liver produces hepcidin. Hepcidin controls iron absorption in the gastrointestinal tract and iron release from reticuloendothelial tissue. Iron must be released from macrophages in the bone marrow to be incorporated into the heme group of hemoglobin in erythrocytes. There are colony-forming units that the cells follow during their formation. These cells are referred to as the committed cells including the granulocyte, monocyte and colony forming units.

1.1.3 MEMBRANE COMPOSITION OF ERYTHROCYTE

The erythrocyte membrane is composed of three major structural elements: a lipid bilayer primarily  composed  of  phospholipids  and  cholesterol  that  provides  a  permeability  barrier between the external environment and the red cell cytoplasm; integral proteins embedded in the lipid bilayer that span the membrane; and a membrane skeleton on the internal side of the red cell membrane that provides structural integrity to the cell. Half of the membrane mass in human and  most  mammalian  red  blood  cells  are  proteins.  The  other  half  are  lipids,  namely phospholipids  and  cholesterol  (Yazdanbakhsh  et  al.,  2000).  The RBC membrane content  is similar with most of animal membranes and it is composed of 19.5% (w/w) of water, 39.5% of proteins, 35.1% of lipids and 5.8% of carbohydrates (Sofia and Saldanha, 2010). The RBC shape is ultimately determined by membrane proteins, especially spectrin network, and also by the lipid bi-layer content (Yawata, 2003). The RBC is able to maintain its discoid shape, and yet allowing cytoskeleton rearrangements that permit it to pass through capillaries, and then restore again its normal shape without cell fragmentation (Yawata, 2003; Pasini et al., 2006). Considerably, lipids are crucial in the maintenance of RBC shape.

1.1.3.1 MEMBRANE PROTEINS

The proteins of the membrane skeleton are responsible for the deformability, flexibility and durability of the red blood cell, enabling it to squeeze through capillaries less than half the diameter of the red blood cell (7–8μm) and recovering the discoid shape as soon as these cells stop receiving compressive forces, in a similar fashion to an object made of rubber.

There are currently more than 50 known membrane proteins, which can exist in a few hundred up to a million copies per red blood cell. Approximately 25 of these membrane proteins carry the various blood group antigens, such as the A, B and Rh antigens, among many others. These membrane proteins can perform a wide diversity of functions, such as transporting ions and molecules across the red cell membrane,  adhesion and interaction with other cells such  as endothelial cells, as signaling receptors, as well as other currently unknown functions. The blood types of humans are due to variations in surface glycoproteins of red blood cells. Disorders of the proteins   in   these   membranes   are   associated   with   many   disorders,   such   as   hereditary

spherocytosis, hereditary elliptocytosis, hereditary stomatocytosis and paroxysmal nocturnal hemoglobinuria(Mohandas and Gallagher, 2008).

1.1.3.2 MEMBRANE LIPID

The human red blood cell (RBC) repeatedly undergoes large elastic deformations when passing through narrow blood vessels. The large flexibility of the RBCs is primarily attributable to the cell membrane, as there are no organelles and filaments inside the cell. The RBC membrane is essentially a two-dimensional (2D) structure, comprised of a cytoskeleton and a lipid bilayer, tethered together. The lipid bilayer includes various types of phospholipids, sphingolipids, cholesterol, and integral membrane proteins, such as band-3 and glycophorin. The RBC membrane resists bending but cannot sustain in-plane static shear stress as the lipids and the proteins diffuse within the lipid bilayer at equilibrium (Tse and Lux, 1999).

The cytoskeleton plays a major role in the integrity of the RBC membrane, as is evident in blood disorders where defects in membrane proteins lead to membrane loss and reduced mechanical robustness of the RBC (Eber and Lux, 2004). In hereditary spherocytosis (HS), the tethering of the cytoskeleton to the lipid bilayer is partially disrupted resulting in membrane loss and subsequently in the spherical shape of the RBCs (Mohandas, 2008).

1.2 HEMOGLOBIN

Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates with the exception of the fish family Channichthyidaeas well as the tissues of some invertebrates (Sidell and Kristin, 2006). Hemoglobin in the blood carries oxygen from the lungs or gills to the rest of the body (i.e. the tissues). There, it releases the oxygen to permit aerobic respiration to provide energy to power the functions of the organism in the process called

metabolism. A healthy individual has “12 to 16” grams of hemoglobin in every 100 ml of blood.

In mammals, the protein makes up about 96% of the red blood cells’ dry content (by weight), and around 35% of the total content (including water). Hemoglobin has an oxygen-binding capacity of 1.34mL O2 per gram, which increases the total blood oxygen capacity seventy-fold compared

to dissolved oxygen in blood (Dominguez et al.,1981). The mammalian hemoglobin molecule can bind (carry) up to four oxygen molecules (Constanzo, 2007).

Hemoglobin is involved in the transport of other gases: It carries some of the body’s respiratory carbondioxide (about 20–25% of the total) as carbaminohemoglobin, in which CO2  is bound to the globin protein. The molecule also carries the important regulatory molecule nitric oxide bound to a globin protein thiol group, releasing it at the same time as oxygen (Epstein and Hsia, 1998).

1.2.1 HEMOGLOBIN SYNTHESIS

Hemoglobin (Hb) is synthesized in a complex series of steps. The heme part is synthesized in a series of steps in the mitochondria and the cytosol of immature red blood cells, while the globin protein parts are synthesized by ribosomes in the cytosol. Production of Hb continues in the cell throughout  its  early  development  from  the  proerythroblast  to  the  reticulocyte  in  the  bone marrow. At this point, the nucleus is lost in mammalian red blood cells, but not in birds and many other species. Even after the loss of the nucleus in mammals, residual ribosomal RNA allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the vasculature (this hemoglobin-synthetic RNA in fact gives the reticulocyte its reticulated appearance and name).

1.2.2HEMOGLOBIN STRUCTURE

Hemoglobin has a quaternary structure characteristic of many multi-subunit globular proteins. Most of the amino acids in hemoglobin form alpha helices, and these helices are connected by short non-helical segments. Hydrogen bonds stabilize the helical sections inside this protein, causing attractions within the molecule, which then causes each polypeptide chain to fold into a specific  shape  (Van  et  al.,  2003).  Hemoglobin’s  quaternary  structure  comes  from  its  four subunits in roughly a tetrahedral arrangement.

In most vertebrates, the hemoglobin molecule is an assembly of four globular protein subunits. Each subunit is composed of a protein chain tightly associated with a non-protein prostheticheme group.  Each  protein  chain  arranges  into  a set  of  alpha-helix  structural  segments  connected

together in a globin fold arrangement. Such a name is given because this arrangement is the same folding motif used in other heme/globin proteins such as myoglobin (Steinberg, 2001). This folding pattern contains a pocket that strongly binds the heme group.

A heme group consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring, known as a porphyrin. This porphyrin ring consists of four pyrrole molecules cyclically linked togetherwith the iron ion bound in the center.The iron ion, which is the site of oxygen binding, coordinates with the four nitrogen atoms in the center of the ring, which all lie in one plane. The iron is bound strongly (covalently) to the globular protein via the N atoms of the imidazole ring of F8 histidine residue (also  known as the proximal histidine) below the porphyrin ring.  A sixth position can reversibly bind oxygen by a coordinate covalent bond, completing the octahedral group of six ligands. Oxygen binds in an “end-on bent” geometry where one oxygen atom binds to Fe and the other protrudes at an angle. When oxygen is not bound, a very weakly bonded water molecule fills the site, forming a distorted octahedron.

The  iron  ion  may  be  either  in  the  Fe2+    or  in  the  Fe3+    state,  but  ferric  hemoglobin (methemoglobin) (Fe3+) cannot bind oxygen (Linberg et al., 1998). In binding, oxygen temporarily and reversibly oxidizes (Fe2+) to (Fe3+) while oxygen temporarily turns into the superoxide ion, thus iron must exist in the +2 oxidation state to bind oxygen. If superoxide ion associated to Fe3+  is protonated, the hemoglobin iron will remain oxidized and incapable of binding oxygen. In such cases, the enzyme methemoglobin reductase will be able to eventually reactivate methemoglobin by reducing the iron center.

In adult humans, the most common hemoglobin type is a tetramer (which contains four subunit proteins) called hemoglobin A, consisting of two α and two β subunits non-covalently bound, each made of 141 and 146 amino acid residues, respectively. This is denoted as α2β2. The subunits are structurally similar and about the same size. Each subunit has a molecular weight of about  16,000 daltons,  for  a  total  molecular  weight  of  the  tetramer  of  about  64,000 daltons (64,458 g/mol) (Van et al., 2001). Hemoglobin A is the most intensively studied of the hemoglobin molecules.

In human infants, the hemoglobin molecule is made up of 2 α chains and 2 γ chains. The gamma chains are gradually replaced by β chains as the infant grows.The four  polypeptide chains are bound to each other by salt bridges, hydrogen bonds, and the hydrophobic effect (Hardison,

1996).

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