PHYTOCHEMICALS ANTIOXIDANTS AND TOXICOLOGICAL PROPERTIES OF METHANOL EXTRACT OF SECURIDACA LONGEPEDUNCULATA

Abstract

One of the problems  associated  with medicinal  plants in Nigeria is paucity of information  on  the phytochemistry  and  toxicity  of  some  of  these  plants.  The  study  aimed  at  revealing  a  range  of phytochemicals  in the plant, which are physiologically  potent in ameliorating  several diseases. The potent secondary metabolites in the leaves of this plant Securidaca longepedunculata , were extracted and their antioxidant  and toxicological  potentials were evaluated using in vitro methods and albino rats  as  the  models.  The    results  showed  that     methanol  extract  scavenged  1,  1-  diphenyl-2- picrylhydrazyl  radical (DPPH.)  in a  concentration  dependent  manner  with a correlation  coefficient (R2) of 0.976, indicating antioxidant activity with effective concentration that inhibits 50 percent of the radicals (EC50) of 90.02±0.2 µg/ml compared to ascorbic acid standard with EC50    of 98.01±0.2 µg/ml.   Superoxide  radical  scavenging   activity  was  concentration   dependent   with  an  EC50    of

350.11±0.42  µg/ml  compared  with  ascorbic  acid standard  with EC50   of 812.97±0.97  µg/ml.  The extract, also showed hydroxyl radical scavenging activity with an EC50 of 83.74±0.02µg/ml compared to α- tocopherol standard with EC50  of 54.16±0.01 µg/ml. There was an inverse correlation between the percentage inhibition and concentration  with R2 of   -0.958. The methanol extract, also scavenged nitric oxide radical in a concentration dependent manner with 500 µg/ml  being more effective  than

500 µg/ml of ascorbic acid standard. Comparison of the anti-radical power (ARP) of DPPH.  (0.011), superoxide radical (0.003) and hydroxyl radical (0.012) of the extract revealed that the ARP of the extract against hydroxyl radical was most efficacious. The antioxidant vitamin contents of the extract showed that vitamin  C was significantly  high (p< 0.05),  (4.62±0.14  mg/100g)  when  compared  to

vitamin A (0.902±0.05 µg/g) and vitamin E (1.474±0.01 mg/100g). The 100, 200 and 500 mg/kg bw fed to rats significantly increased (p< 0.05) catalase activity, while in weeks two and three the catalase activity  decreased  significantly  (p< 0.05).  The extract  solution  showed  a maximum  absorption  at wavelength  (lambdamax)  of 285nm-thus  indicating  that  subsequent  investigations  using the extract would be better at UV region in absorption spectra. There was no death in the mean lethal dose (LD50) investigation. The aspartate aminotransferase (AST) showed a significant decrease (p< 0.05) in week one  and  an  increase  in  other  weeks.  The  alanine  aminotransferase  (ALT)  showed  a  significant decrease (p< 0.05),   in weeks one, three and four while week two showed a non-significant increase (p> 0.05). The serum alkaline phosphatase  (ALP) showed a significant (P   0.05) decrease in activity in all the groups at weeks one and two. At week three only group two showed a non significant increase (P   0.05); others showed a non significant decrease (P  0.05). At week four, all the groups showed an increase, but only groups two and three were significant (P

0.05) when compared to their controls group 1. Both conjugated and unconjugated bilirubin of groups two to four of weeks two to four showed generally a significant increase (p< 0.05),  compared with that of the control. The serum sodium level showed a significant increase (p< 0.05), in groups two and three of week one. Weeks two to three showed a non-significant increase (p> 0.05) compared to that of the control group. Serum potassium and chloride ion concentrations  showed a significant decrease (p< 0.05)   in weeks one to three and significant increase (p< 0.05) in week four.  The serum urea showed overall significant decrease (p< 0.05) compared to that of the control group (p< 0.05). The serum creatinine showed a significant increase (p< 0.05) in weeks one and two, a non-significant decrease (p> 0.05)  in week three  and a significant decrease in week four (p< 0.05). Weeks one and two rats showed significant decrease (p< 0.05) in random blood sugar (RBS), while in weeks three and four, the rats showed significant increase (p< 0.05) in RBS concentration. The packed cell volume (PCV) and  haemoglobin  concentration  (Hb) showed significant  decrease (p< 0.05) when compared with those of the control group. The white blood cell (WBC) counts of the rats showed significant increase (p< 0.05). Histological  analysis showed some level of toxicity in 100, 200 and 500  mg/kg b.w. at chronic stage (beyond  14 days of administration).  These results  seemed  to  suggest  a rich phytochemical constituents, suggested a moderate antioxidant activity, a relatively safe level at acute phase (within 14 days) and a visible damage (moderate toxicity) at chronic stage.

CHAPTER ONE

INTRODUCTION

1.1 Introduction

Phytomedicinal  research of indigenous plant parts is presently gaining more grounds  than ever  as the majority of people  are now patronizing  herbal  medicinal  treatment  which  is considered  to  be  more  easily  accessible  and  cheaper  than  orthodox  medical  treatment (Ajiboye et al., 2010). The usefulness of medicinal plants is directly linked to the wide range of chemical compounds synthesized in various biochemical pathways; which are classified as secondary metabolites(Ameyawand Duker-Eshun, 2009). The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body (Edoga  et  al., 2005;  Lavanya  et  al.,  2007).  Antioxidants  can  delay or  inhibit  the oxidation  of  lipids,  proteins,  DNA  or  other  biomolecules  by inhibiting  the  initiation  or propagation of oxidative chain reaction, (Awah et al., 2010), thereby preventing the untimely death of a cell and some ailments. The measurement of enzyme activities in the body fluid aids the diagnosis of assault on organs and tissues and also assists the determination of the toxicity of chemical compounds or drugs (Ajiboye et al., 2010).

1.2Profile ofSecuridaca longependunculata

Securidaca longepedunculata Linn belonging to the family of polygalaceae, is a spiny semi- deciduous shrub that grows to about 12m tall (Owoyele et al., 2006). It is widely distributed in West African and South African regions (Abdullahi and Lawal, 2010). The plant is known as Rhodes’  violet  wild  vesteria  (English),  uwar magunguna  (Hausa)  and  Atumaka  (Ibo). Eaten  as  vegetable,Securidaca   longepedunculata  was  reported  to  have  analgesic,  anti- inflammatory and hypoglycemic potentials (Ajiboye et al., 2010), and has high efficacy in treating  malaria  and  rheumatism(Gill,  1992).  It  is  also  used  in  bacterial  chemotherapy (Akinniyi et al., 1996; Mohammed et al., 2012). The plant is also used in the treatment of cough, headache, constipation, wound, sore throat and gout (Ojewale, 2008). Its anti-snake venom potential has also been reported (Wannag et al., 2005). Some secondary metabolite contents such as alkaloids and flavonoids and related compounds such as methyl salicylate has been reported (Iddagoda and Thamara, 2003;Abdullahi and Lawal, (2010).

Fig.1Securidaca longepedunculata leaf and flower

1.2.1Phytochemistry

The  significance  of  medicinal  plants  is  directly  linked  to  the  wide  range  of  chemical compounds   synthesized   in  the  various  biochemical   pathways.   These   compounds   are classified  as secondary  metabolites  (Ameyaw  and  Duker-Eshun,2009).The  importance  of natural molecules  in medicine  lies  not  only in  their  pharmaceutical  or chemotherapeutic effects but also in their role as template molecules for the production of synthetic drugs. Most anti-malarial  drugs  currently  in  use  are  quinoline  derivatives  modeled  on  the  quinine molecule.  (Ameyaw  and  Duker-Eshun,  2009). There  are three large classes of secondary metabolites  in pants:  Nitrogen-containing  compounds,  terpenoids  and phenolics.  Some of these phytochemicals are alkaloids, flavonoids, saponin, resins, tannins, among others.

1.2.2 Alkaloids

Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms. They have pharmacological  effect and biological activities  such as anti-malarial,  anti-microbial,

anti-hyperglycemic  and anti-inflammatory  effect and are used as medications,  recreational drugs, or in etheogenic rituals (Tackie and Schiff, 1993). There is, also the non-basic forms such  as quaternary  compounds  and  N-oxides  (Ameyaw  and  Duker-Eshun,  2009).  Many alkaloids are poisonous to other organisms.

1.2.3 Flavonoids

Flavonoids,  carotenoids  among others are among the antioxidants  produced  by plants  for their own sustenance (Ali et al., 2010). Flavonoids have hydroxyl group (OH). The effect of hydroxyl moiety of flavonoids on protein targets varies depending on position and number of the moiety on the flavonoid skeleton (Hyunchu et al.,2010).

1.2.4 Tannin

Tannins are naturally occurring plant polyphenols. They bind and precipitate proteins. They form complexes, also with carbohydrates, bacterial cell membranes and enzymes involved in protein and carbohydrate digestion. The tannin phenolic group is an excellent hydrogen donor that forms strong hydrogen bonds with the protein’s carboxyl group (Reed, 1995). The anti carcinogenic  and anti mutagenic  potentials of tannins may be  related  to their antioxidant property (Chung et al., 1998). The anti-microbial properties seemed to be associated with the hydrolysis of ester linkage between gallic acid and polyols hydrolyzed after ripening of many edible fruits (Chunget al., 1998).

1.2.5 Total Phenolics

Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids (Kahkonen et al., 1999). Antioxidant activity of plant extracts is not limited to phenolics.  Activity  may  also  come  from  the  presence  of  other  antioxidant  secondary metabolites,  such as volatile oils, carotenoids and vitamins A, C  and E. Crude extracts of fruits, herbs, vegetables, cereals and other plant materials rich in phenolics are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food  (Shahidi et al., 1992). Phenolics are the main antioxidant components of food. While in plants, oils are basically monophenolics such as tocopherols,  water-soluble  polyphenols  are more typical in water-soluble  products  like fruits, vegetables, tea, coffee, wine, among others (Roginsky and Lissi, 2004). Polyphenolic compounds are known to have antioxidant activity. This activity is believed to be mainly due

to their redox properties which play an important  role in adsorbing and neutralizing  free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Ali et al., 2010).

1.3 Acute Toxicity

Acute toxicity describes the adverse effects of a substance that result either from a  single exposure or from multiple exposures in a short space of time (less than 24 hours). Most acute toxicity data come  from  animal  testing  or  in  vitro  testing  methods  (Walum,  1998).  The median lethal dose (LD50) is the dose required to kill half the members of a tested population after a specified test duration. LD50 figures are  frequently used as a general indicator of a substance’s   acute   toxicity.   LD50    is   dependent   on  the   gene,   animal   species   tested, environmental factors and mode of administration (Ernest, 2004).

1.4 Reactive Oxygen Species

Reactive  oxygen  species  (ROS),  which consist  of free radicals  such  as  hydroxyl  radical (OH.), superoxide  radical (O2-), Nitric oxide (NO-), peroxyl (RO2-), lipid  peroxyl  (LOO-) radicals and non-free radical species such as hydrogen peroxide (H2O2), singlet oxygen (O2-

1), ozone (O3), lipid peroxide (LOOH), are different forms of activated oxygen (Halliwell and

Gutteridge,  1999; Yildrin and Mavi, 2000; Gulcin et al.,2004).  ROS are produced  by all aerobic organisms and can easily react with most biological molecules including  proteins, lipids, lipoproteins and DNA, leading to an imbalance between the systemic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify or repair the resulting damage (Halliwell, 2007). During times of  environmental stress, ROS levels can increase  dramatically.  These ROS can also  generate  oxidative  stress. Cumulatively,  ROS could lead to many pathophysiological  disorders such as arthritis, diabetes, inflammation, cancer and genotoxicity (Kourounakis and Galanakis, 1999; Gulcin and Oktay, 2002).

Experiments  show  that  ROS  induce  chronic  inflammation  by  the  induction  of  COX-2 inflammatory cytokines. These chemokines and their receptors, in turn, promote invasion and metastasis of various tumour types (Gupta et al., 2012). ROS have  important roles in cell signaling  and homeostasis  (Davasagayam  et al., 2004).  Reactive  oxygen  species  such as superoxide  anions, hydroxyl radical, nitric oxide  radical among others inactivate enzymes and damage the important cellular components causing injury through covalent binding and lipid  peroxidation.  Generation  of hydroxyl  radicals is crucial for the  irreversible  damage

inflicted by oxidative stress (Halliwell& Gutteridge, 1999). This generation mainly proceeds

via Fenton reaction:

H2O2 + Fe2+ → Fe3+ + HO– + HO•, (1)

as well as in reaction between hypochlorous acid and superoxide anion: HOCl + O2– → O2 + Cl– + HO•    (2)

The rate constant of the latter reaction is greater than that of the reaction of Fe2+ with H2O2.

Decomposition of peroxynitrous acid also yields HO•: HONOO → NO2 + HO• (3)

This reaction seems to be responsible for some 20-30 % of the decay of peroxynitrite. The hydroxyl radical is an extremely reactive species and reacts at a high rate (k ~ 109–1010 M–1 · s–1) with all surrounding molecules: proteins, lipids, nucleic acidsand  sugars. Because the hydroxyl radical recombinationHO•  +•OH → H2O2 is also very fast (k = 5 × 109 M–1 · s–1) the steady-state  concentration  of  hydroxyl  radical  is practically  zero  (Halliwell&  Gutteridge,

1999).  Nitric  oxide  (NO.)  has  also  been  involved  in  a  variety  of  biological  functions, including neurotransmission, vascular homeostasis, antimicrobial, and antitumor activities.

Despite the possible beneficial effects of NO., its contribution to oxidative damage is also reported. This is due to the fact that NO.can react with superoxide to form the peroxynitrite anion, which is a potential oxidant that can decompose to produce OH.and NO.  (Rajesh and Natvar,2011). In cellular oxidation reactions, superoxide radicals are  normally formed and

their  effects  can  be  magnified  because  they  produce  other  kinds  of  cell-damaging  free radicals and oxidizing agents. Superoxide  is biologically  important  as it can form  singlet oxygen and hydroxyl  radical.  Over production of superoxide  anion radical  contributes  to redox imbalance, associated with harmful physiological consequences  (Rajesh and Natvar,

2011).

1.5.1 Antioxidants

Antioxidants are substances that, when present in low concentrations, compared to that of an oxidizable substrate, significantly delay or prevent oxidation of that substance (Halliwell and Gutteridge,  1989).Two  classes of antioxidants  are known: (1). The low  molecular  weight compounds such as vitamins E and C, beta-carotene, glutathione, uric acid, bilirubin, among others  and  (2).The  proteins:  albumin,   transferrin,   caeruloplasmin,   ferritin,   superoxide dismutase, glutathione peroxidase, catalase, among others (Psotova et al., 2001).   Typical phenolics  that  possess  antioxidant  activity  have  been characterized  as phenolic  acid  and

flavonoids  (Kahkonen  et  al.,  1999).  The  increasing  awareness  of  consumers  to  issues regarding food additive safety, results in an enhanced effort in finding alternative additives and preservatives from natural and probably safer sources. The main disadvantage with the synthetic   antioxidants   is  their  side  effects  when  taken  in   vivo.In  this  regard,   food manufacturers  have  been  encouraged   to  use  natural   antioxidants   instead  of  synthetic compounds  to maintain the nutritional  values of  their products. For example, commercial antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) can  be  replaced  by  plant  extracts,  particularly  polyphenols  (Balasundram  et  al.,  2006). Antioxidants are added to  prevent  or delay its (food) oxidation,  normally initiated by free radicals formed during  the food’s exposure to environmental factors such as air, light and temperature (Hras et al., 2000). Generation of free radicals or reactive oxygen species (ROS) during  metabolism  and  other  activities  beyond  the  antioxidant  capacity  of  a  biological system, gives rise to oxidative stress. Oxidative stress (OS) plays a role in heart  diseases, neurodegenerative diseases, cancer and in aging process (Astley, 2003). Antioxidants oppose these effects, thus lowering the risk of diseases (Atouiet al., 2005).

1.5.2 Antioxidant Vitamins

Vitamins are compounds that are essential to the health of humans and vertebrates but cannot be synthesized by these animals and must therefore be obtained in the diet (Nelson and Cox,

2005). Vitamin C, E and Beta-carotene are among dietary antioxidants, (Sies, 1992). Vitamin C is considered  the most important  water soluble antioxidant  in extracellular  fluids. It is capable of neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Sies,

1992). Vitamin C is capable  of regenerating  vitamin E. Vitamin E, a major  lipid-soluble antioxidant is the most effective chain-breaking antioxidant within the cell membrane where it protects membrane fatty acids from lipid peroxidation. Low intakes of dietary antioxidants are  linked  to  an  increased  frequency  of  heart  disease  (Hennekens  and  Gaziano,  1993). Antioxidants have been shown to prevent low density lipoprotein (LDL) oxidation in vitro and retard the progression of atherosclerosis in animal models. Several human studies found supplemental  vitamin  E  levels  in  LDL,  increased  the  resistance  of  LDL  oxidation  and decreased the rate of LDL oxidation; LDL oxidation is speculated in atherosclerosis (Jialal and  Fuller,  1993).   Beta-carotene   and  other  carotenoids  are  also  believed  to  provide antioxidant  protection  to  lipid-rich  tissues.  It  may  work  synergistically  with  vitamin  E, (Jacob, 1995). By extension, a diet that is excessively low in fat may negatively affect beta- carotene and vitamin E absorption, as well as other fat-soluble nutrients.

1.5.3 Catalase

Catalase is an enzyme found in nearly all living organisms exposed to oxygen. It catalyzes the decomposition of hydrogen peroxide to water and oxygen (Chelikani et al., 2004)

catalase

2H2O2                                     2H2O + O2

It is very important in protecting the cell from oxidative damage caused by reactive oxygen species (ROS), (Goodsell, 2004). Catalase is a tetramer of four polypeptide chains, each over

500 amino acids long (Boon et al., 2007). It contains four porphyrin heme (iron) groups that allow  the  enzyme  to  react  with  hydrogen  peroxide,  (Maehly  and  Chance,  1954).  The reactivity of the iron centre may be improved by the presence of the phenolate ligand of Tyr

357 in the fifth iron ligand which facilitates the reduction of hydrogen peroxide, (Boon et al.,

2007).

1.6 1,1-Diphenyl-2-picrylhydrazyl  radical(DPPH.) Assay

Fig. 2 Structures of 1,1-diphenyl-2-picrylhydrazyl radical and 1,1-diphenyl-2-picrylhydrazine

Source: Sagar and Singh, 2011

This 1,1- Diphenyl-2-PicrylHydrazyl  radical (DPPH.), (Fig. 2) is characterized  as a  stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that the molecules  do not dimerise  like most other free radicals.  The  odd electron of

nitrogen atom in DPPH.is reduced by receiving a hydrogen atom from antioxidants to  the

corresponding hydrazine, (Sagar and Singh, 2011). The delocalization also gives rise to the

deep  violet  colour,  with  an  absorption  in  ethanol  solution  at  515-  520nm.  On  mixing DPPH.solution with a substance that can donate a hydrogen atom, it gives rise to the reduced form with the loss of violet colour. It thus undergoes a barthochromic shift (which is a shift from a colour of shorter wavelength to a colour of longer wavelength) (Akpanisi, 2004).

1.7.1 Liver Function Tests

The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum can help diagnose the injury of body tissues or organs especially the heart and  the liver (Huang et al., 2006). AST and ALT are enzymes found mainly in the liver, but also found in red blood cells, heart cells, muscle tissue and other organs, such as pancreas and kidney. The normal concentrations in the blood are from 5 to 40 U/L (unit/litre) for AST and from 5 to 35

U/L for  ALT.  When body tissues  or an organ  such as the  liver  or heart  is diseased  or damaged, additional AST and ALT are released into the blood stream, causing the levels of the enzyme to rise. Therefore, the amount of AST and ALT in the blood is directly related to the extent of the tissue damage (Sampson et al.,1980). Alkaline Phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in an alkaline environment, resulting in the formation of an organic radical and inorganic phosphate. In  mammals, this enzyme is found mainly in the liver   and   bones.   Marked   increase    in   serum   ALP   levels,   a   disease   known   as hyperalkalinephosphatasemia,   has   been   associated   with  malignant   biliary  obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and sarcoidosis (Kim, 2006).

1.8.0 Kidney Function Test

Renal function is an indication of the state of the kidney and its role in renal  physiology (Stevens et al., 2006). Creatinine clearance rate is the volume of blood plasma that is cleared of creatinine per unit time and is a useful measure for approximating Glomerular Filtration Rate (GFR). Blood urea nitrogen and creatinine will not be raised  above the normal range until 60% of total kidney function is lost. So there is the need to check creatinine clearance rate whenever kidney disease is suspected. A high creatinine level points to kidney disease. Low urea nitrogen concentrations  were  alluded  to over hydration,  starvation,  and alcohol (Gifford and Leel-Khouri,  1989).  Low urea can also be due to urea cycle defects. Serum creatinine is better than urea in assessing GFR.

1.8.1 Serum Electrolytes

1.8.1.1 Sodium

Sodium is the dominant extracellular cation (positive ion) and cannot freely cross from the interstitial  space  through  the  cell  membrane,  into  the  cell.  Its  homeostasis  (stability  of concentration) inside the cell is vital to the normal function of any cell. Hyponatremia is low sodium concentration in the serum. Exercise can induce hyponatremia. When sodium levels in the blood become excessively low, excess water enters the brain cells and the cells swell- this can lead to headache, nausea, vomiting confusion and seizures (Moritz and Ayus, 2003). The main source of body sodium is sodium chloride contained in ingested foods (Terri and Sesin,  1958).  Hyponatremia  is  found  in a variety of conditions  including  the  following: severe polyuria, metabolic  acidosis, Addison’s disease, diarrhoea and renal tubular disease. Hypernatremia   (increased   serum  sodium  level)  is  found  in  the  following  conditions: hyperadrenalism,   severe  dehydration,  diabetic  coma  after  therapy  with  insulin,  excess treatment with sodium salts (Trinder, 1951 Maruna, 1958).

1.8.1.2 Potassium

Potassium,  a  metallic  inorganic  ion,  is the  most  abundant  cation  in the  body.  The  vast majority  of  potassium  is  in  the  intracellular  compartment  with  a  small  amount  in  the extracellular  space. While total body potassium is lower in females and in  older  patients, serum potassium concentration is independent of age and sex (Adrogue and Madias, 1981). Total body potassium is approximately 55mEq/Kg body weight. The intracellular potassium

concentration is on average 150mEq/L. The ratio of intracellular to extracellular K+      (k1:k2)

is the major determinant of the resting membrane potential and plays a crucial role in the normal  functioning  of all cells,  especially  those  with  inherent  excitability  (Aruda  et al.,

1981). Elevated potassium (hyperkalemia) is often associated with renal failure, dehydration, shock or adrenal insufficiency. Decreased potassium levels in the plasma (hypokalemia) are associated  with  malnutrition,  negative  nitrogen  balance,  gastrointestinal  fluid  losses  and hyperactivity of adrenal cortex (Terri and Sesin, 1958).

1.8.1.3 Chloride

An abnormal elevation of chloride ion concentration in the blood is hyperchloremia.  It  is associated  with excess  fluid  loss such as vomiting and diarrhoea.  Diabetes  exacerbates  it (Cambier  et  al.,  1998).  Non-  steroidal  anti-inflammatory  drugs  can  modulate  chloride

concentration in the blood. Chloride is the major negative ion in the fluid outside the body’s cells. Its main function is to maintain electrical neutrality, mostly as a counter-ion to Sodium (Terri and Sesin, 1958). It is therefore important to the control of proper hydration, osmotic pressure  and acid/base  equilibrium.  Low serum Chloride values are  found with extensive burns, excessive vomiting, intestinal obstruction, nephritis and metabolic acidosis. Elevated serum Chloride values may be seen in dehydration, hyperventilation, congestive heart valve and prostatic obstruction (Skeggs and Hochstrasser, 1964).

1.9 Haematology

Packed cell volume (PCV) or erythrocyte volume fraction (EVF), is the volume percentage (%) of red blood cells in blood. It is normally about 45% for men and  40% for women (Purves, et al., 2004). It is considered an integral part of a person’s complete blood count results,  along with haemoglobin  concentration,  white blood cell  count and platelet count. Erythropoietin is secreted by the kidney. So, by extension, low level of red blood cells points to a problem with the integrity of the kidney cells (Jelkmann, 2004). Haemoglobin is the iron- containing oxygen-transport metalloprotein in the blood cells of almost all the vertebrates. It carries oxygen to body parts for the metabolism of glucose in order to generate energy (Sidell and Kristin, 2006). If haemoglobin is low, it is called anemia. Anemia is a condition in which the  number  of   red  blood  cells  (and  consequently  their  oxygen-carrying   capacity)  is insufficient to meet the body’s needs, (WHO, 2001). Sickle cell anemia is the most important hemoglobinopathy, (Murray et al., 2006). Residential elevation above sea level and smoking are known to increase haemoglobin concentrations, (WHO, 2001).

1.10 Histopathology

Histopathology   refers   to   microscopic   examination   of   tissue   in  order   to   study   the manifestation  of  disease.  Histopathological  examination  starts  with  surgery,   biopsy  or autopsy.  Fixative  stabilized  the  tissue  and  thereby  prevented  decay.  The  most  common fixative is formalin (10% formaldehyde in water). The medical  diagnosis is formulated  as pathology report describing the histological findings and the opinion of the pathologist.

1.11.0 Aim and Objectives

1.11.1 Aim

The outstanding  disadvantages  of herbal medicinal  treatment  are that the toxicity of  the concoctions taken by patients is not determined nor the active principles standardized. The main aim of this study is to investigate,in vitro, antioxidant contents of the methanol extract of Securidaca longepedunculata and also to determine the toxicity of this plant widely used in Nigerian folk medicine to cure various ailments.

1.11.2 Objectives

The study was designed to achieve the following specific objectives:

1.   To determine the phytochemical constituents of Securidaca longepedunculata

2.   To determine its antioxidant potential

3.   To    determine    possible    acute    toxicity(LD50)    concentration    of    Securidaca longepedunculata with a view to determining the non-toxic safe dose of the extract.

4.   To determine the effect of the extract on liver marker enzymes and kidney function profile.

5.   Determination  of  the  possible  effects  of  Securidaca  longepedunculata  methanol extract  on  the  kidney  and  liver  cells  of  Wister  male  rats  through  histological technique. 6.   To determine the effect of the extract on blood cells.

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