EFFECT OF THE METHANOL EXTRACT OF JATROPHA TANJORENSIS LEAVES ON SOME HAEMATOLOGICAL AND ANTIOXIDANT PARAMETERS IN RATS

ABSTRACT

The effect of the methanol extract of Jatropha tanjorensis leaves on haematological and antioxidant parameters of rats was investigated. From the study, the percentage yield of the extract  was 9.77%. The qualitative and quantitative phytochemical screening of the extract revealed the presence of active principles such as tannins (69.91±7.53 mg/100 g), flavonoids (3116.15±143.24 mg/100 g), total phenolics (12.98±1.02 mgGAE), steroids (26.17±0.09 mg/100g), terpenoids (208.80±48.31 mg/100 g), alkaloids (1677.78±41.95 mg/100 g), and carbohydrate (1345.29±2.26 mg/100 g). The extract showed no visible signs of toxicity even at a dose of 5000 mg/kg body weight. Twenty-eight days repeated dose toxicity was carried out using twenty-four rats divided into four groups of six rats each. The animals in group 1 served as control and received distilled water while groups 2, 3 and 4 received 100 mg/kg, 200 mg/kg and 400 mg/kg body weight of the extract respectively. The results obtained showed that the extract significantly (p  <  0.05) increased the  mean red  blood cell  count,  packed cell  volume and haemoglobin concentration of the animals treated with 200 mg/kg and 400 mg/kg body weight of the extract when compared to the values obtained for the control group. The result also revealed a reduction in the mean white blood cell count in all the treated groups when compared to the values obtained for the control. However, this reduction was significant (p < 0.05) in the animals that received 400 mg/kg body weight of the extract. There was also an increase in platelet count of the treated groups when compared to the value obtained for the control. However, the increase was significant (p < 0.05) in the animals treated with 400 mg/kg of the extract compared with the control. The malondialdehyde (MDA) concentration decreased significantly (p < 0.05) in all the treated groups when compared to the value obtained for the control. Results from some of the antioxidant enzymes studied revealed that the extract increased glutathione peroxidase (GPx) and catalase  (CAT)  activities  of the  treated  groups  when compared to  the  controls.  However, treatment with 200 mg/kg body weight of the extract caused a significant (p < 0.05) increase in superoxide dismutase (SOD) activity when compared to control while the increase was non- significant (p > 0.05) in the other treated groups compared to control. The result showed that apart from the WBC and platelet counts, the highest activity of the extract was obtained at the dose of 200 mg/kg body weight after which a decline was observed at a higher dose. The results obtained from this study might indicate that the methanol extract of Jatropha tanjorensis leaves contained phytochemicals which are capable of improving the haematological and antioxidant

CHAPTER ONE

INTRODUCTION

Proteins, carbohydrates and fats as well as vitamins and minerals are made available to man and other animals through green plants (Iwalewa et al., 2005). Some plants, apart from serving as food, have also been known to possess medicinal properties (Borget, 1993). There are  many plants  including green leafy  vegetables that  have  several  health benefits and nutritional values for mankind (Ozuola et al., 2006). Vegetables are herbaceous plants whose parts are eaten as supporting food or main dishes and they may be aromatic, bitter or tasteless (Mensah et al., 2008). The consumption of leafy vegetable is part of Africans’ cultural heritage and they play important roles in the tradition and food culture of many African households (Mensah et al., 2008). Nigeria is endowed with a variety of indigenous green leafy vegetables which are consumed by various groups for different reasons. Vegetables are the  cheapest  and  most  available  sources  of  important  proteins,  vitamins,  minerals  and essential amino acids (Weiss, 2005). The active ingredients for a vast number of pharmaceutically derived medications contain components originating from phytochemicals (Lewis and  Manony, 1977).  Despite the  effectiveness  of chemically  synthesized drugs, screening plants for drugs has continued for the development of new pharmaceuticals to resolve both old and new health problems (Iwu, 1983). The plant kingdom offers a wide range of natural antioxidants and medicinal plants (Lewis and Manony, 1977).

A number of studies have revealed the antioxidant activities of phytochemical constituents of medicinal plants (e.g.  polyphenols, carotenoids, phenolics and  vitamins C  and  E).  These phytochemicals  acting  as  antioxidants  prevent  damages  to  cell  membrane  and  cellular oxidative processes that may give rise to diseases (Omoregie and Osagie, 2007). For instance, natural polyphenols from plant vegetables have been found to exert their beneficial effect by removing free radicals, chelating metal catalysts, activating antioxidant enzymes (Ayoola et al., 2006). In recent times, antioxidants from plant sources have received a lot of attention and are  even  preferred  to  synthetic  ones  especially  due  to  their  potential  health  benefits, availability, affordability and in many cases, reduced toxicity (Tarawneh et al., 2010).

Although there is growing popularity that herbal medicines are safe, scientists still advocate for proper physiological and toxicological tests in order to ensure safety in their use (Oyewole et al., 2007, Ozuola et al., 2006). The aim of this study was to investigate the effect of methanol

extract of Jatropha tanjorensis leaves on some haematological parameters and antioxidant enzymes of rats.

1.1 Jatropha tanjorensis

The name Jatropha is derived from the Greek iatrós (doctor) and trophé (food) which implies medicinal use (Mousumi and Bisen, 2008). Jatropha tanjorensis is a member of the “Euphorbiacea” family. It is popularly referred to as “Hospital Too Far”, catholic vegetable,

‘Iyana-Ipaja’ or ‘lapalapa’ by the local folks in different parts of Nigeria (Iwalewa et al.,

2005). Other species of this plant are Jatropha curcas, Jatropha gossypifolia, Jatropha podagrica, Jatropha glandulifolia, Jatropha multifida, Jatropha intergerrima (Debnath and Bisen, 2008).Jatropha tanjorensis is a bushy, gregarious shrub of about 1.8 metres in height. The leaves are 3-5 lobed palmate and contain glandular hairs. The white-coloured flowers, which are usually borne on cyme-branched inflorescences, may contain 3-forked arrangements in  which  the  pistillate  flowers  are  located  on  the  basal  fork.  The  staminate  flowers  are expanded distally from the base of the lobes. Mature seeds and fruit are rare and unknown (Harris and  Munsell, 1950). Jatropha species have a  high adaptability for thriving under different climatic conditions (Debnath and Bisen, 2008). It is, therefore, suitable for all types of soils and barren land. Jatropha is a versatile plant owing to its excellent regeneration capability and long, productive life.

1.2.1 Jatropha tanjorensis: Taxonomy

Kingdom                                                     Plantae Phyllum                                                       Tracheophyta Class                                                            Magnoliopsida Order                                                           Malpighiales Family                                                         Euphorbiaceae Genus                                                          Jatropha

Species                                                        Jatropha tanjorensis

(Source: Ellis and Saroja, 1961)

1.2.2 Uses of J. tanjorensis

Different parts of Jatropha plants are used in many ways and in different countries. Nutritionally, the leaves of J. tanjorensis are locally consumed as vegetable (Orhue et al.,

2008; Iwalewa et al., 2005).The leaves also serve medicinal purposes as they are used for the treatment of fevers, cabuncles, eczema, itches, sores on the tongues of babies, stomach ache and  venereal diseases (Oduola, 2005).  In the  southern parts of Nigeria,  the  leaves  of J. tanjorensis are used for the treatment of diabetes mellitus (Olayiwola et al., 2004). It is also popular as a natural remedy against malarial infection and hypertension in some parts of Nigeria (Iwalewa et al., 2005).

1.2.3 Phytochemical analysis of Jatropha tanjorensis

Earlier studies on the J. tanjorensis leaf revealed that it contains bioactive principles such as alkaloids, flavonoids, tannins, cardiac glycosides, anthraquinones, and saponins (Ehimwenma and  Osagie,  2007).  It  also  contains  important  mineral  elements  such  as  iron,  sodium, potassium, calcium, selenium, manganese, magnesium, phosphorus and zinc (Oboh and Masodje, 2009; Idu et al., 2014). According to Omobuwajo et al. (2011), the leaves contain the antioxidant vitamins (vitamins C and E).

1.3 Phytochemicals

Phytochemicals are secondary metabolites produced by plants and they give plants their colour, flavour, smell and are parts of a plant’s natural defence system (Agte et al., 2000). They are present in a variety of plants utilized as important components of both human and animal diets including seeds, herbs, fruits and vegetables (Criagg and David, 2001). Different mechanisms have  been  suggested  for  the  action of phytochemicals.  They  may act  as  antioxidants or modulate gene expression and signal transduction pathways (Doughari et al., 2009).They may be used as chemotherapeutic or chemopreventive agents (D’Incalci et al., 2005). These bioactive compounds, usually present in small quantities in higher plants, include the alkaloids, flavonoids, tannins, terpenoids, steroids and saponins.

1.3.1 Saponins

Saponins are a group of secondary metabolites, nonvolatile surfactants that are widely distributed  in  the  plant  kingdom and  marine  animals  (Vincken  et  al.,  2007).  The  name

‘saponin’ comes from soap. Saponins have a range of properties which include certain features and  are  due  to  their  extensive  structural  diversity.  They  are  used  as  bitter  sweeteners, detergents and they also possess emulsifying properties. In addition to these properties, they have also been shown to exhibit biological, medical and pharmacological activities, such as haemolytic, antimicrobial, insecticidal and molluscicidal activities (Sparg et al., 2004; Barbosa,

2014).

1.3.2 Tannins

Tannins are an exceptional group of water soluble phenolic metabolites of relatively high molecular weight. They have the ability to complex strongly with carbohydrates and proteins (Heldt and Heldt, 2005). They are astringent, bitter plant polyphenols that cause the dry and pucker feeling in the mouth when unripe fruits or red wines are consumed (Serafini et al.,

1994).  Several health  benefits  have  been attributed to  tannins and  some  epidemiological associations with the decreased frequency of chronic diseases have been established (Serrano et al., 2009). Several studies have shown significant biological effects of tannins such as antioxidant or free radical-scavenging activity,  as well as,  inhibition of lipid peroxidation (Amarowicz et al., 2000). They have also been shown to possess anti-microbial, anti-viral, anti-mutagenic and anti-diabetic properties (Gafner et al., 1997). The antioxidant activity of tannins has been suggested to be a result of their free radical-scavenging properties as well as chelation of transition metal ions that modify the oxidation process (Serrano et al., 2009).

1.3.3 Phenolics

Phenolic compounds are a group of chemical compounds that are widely distributed in nature. Plant phenolics are generally involved in defence against ultraviolet radiation or aggression by pathogens, parasites and predators, as well as contributing to plants’ colours (Kondo and Kawashima, 2000). They are ubiquitous in all plant organs and are therefore an integral part of the human diet (Boudet, 2007). Phenolics are widespread constituents of plant foods (such as fruits, vegetables, cereals, olive, legumes and chocolate) and beverages (such as tea, coffee, beer and wine), and partially responsible for the overall organoleptic properties of plant foods

(Heo  et  al.,  2004).  Phenolic  compounds possess  different  biological activities,  but  most important are antioxidant activities (Rice-Evans, et al., 1997). Researchers and food manufacturers have become more interested in polyphenols due to their potent antioxidant properties, their abundance in the diet, and their credible effects in the prevention of various oxidative stress associated diseases (Manach et al., 2004). Phenolics are able to scavenge reactive oxygen species due to their electron-donating properties. Their antioxidant effectiveness in food depends not only on the number and location of hydroxyl groups but also on   factors   such  as   physical   location,   interaction  with   other   food   components,  and environmental conditions (e.g., pH) (Re et al., 1999).

1.3.4 Terpenoids

Terpenoids, also known as isoprenoids are the major family of natural compounds comprising more than 40,000 different molecules (McCaskill and Croteau, 1998). The isoprenoid biosynthetic  pathway produces both primary and  secondary metabolites that  are  of great significance to plant growth and persistence (Haudenschild and Croteau, 1998). Many of the terpenoids are important for the quality of agricultural products such as the flavours of fruits and the fragrance of flowers like linalool (Pichersky et al., 1994). In addition, terpenoids have medicinal properties such as anti-carcinogenic (e.g. taxol and perilla alcohol), antimalarial (e.g. artemisinin), anti-ulcer, anti-microbial or diuretic activity (e.g. glycyrrhizin) (McCaskill and Croteau, 1998; Bertea et al., 2005).

1.3.5 Flavonoids

Flavonoids are  polypenolic compounds that  are  ubiquitous in  nature  and  are  categorized according to their chemical structures into flavones, anthocyanidins, isoflavones, catechins, flavonols, chalcones and  flavonones (Williams  and  Grayer,  2004).  They occur mostly  in vegetables, fruits and beverages like tea, coffee and fruit drinks. They accumulate in plants as phytoalexins defending them against microbial attack (Grayer and Harborne, 1994) and fungal attack (Jensen et al., 1998).These plant metabolites have been shown to possess useful biological effects on humans including anti-inflammatory activity, enzyme inhibition and anti- microbial  activity  (Havsteen,  1983;  Harborne  and  Williams,  2000),  antioxidant  and  free radical-scavenging ability (Robak and Gryglewski, 1988). They have also  been shown to

possess mild vasodilatory properties useful for the treatment of heart diseases (Hodek et al.,

2002).

1.3.6 Alkaloids

Alkaloids are groups of naturally occurring chemical compounds that are mainly of plant origin and contain nitrogen usually derive from amino acids. In plants over 12,000 alkaloids are known and several are used medicinally (Mattijs et al., 2006). They are also one of the most diverse groups of secondary metabolites found in living organisms. In seeds and insects, toxic alkaloids  are  sequestered for  use  as  a  passive  defence  agent  by  acting  as  deterrents for predating insects. Many of the plants that contain alkaloids are medicinal plants and have been used as herbs. Examples of such alkaloids include aconitine, atropine, ergotamine, morphine, ephedrine, strychnine, psilocin and psilocybin (Schmeller and  Wink, 1998). Alkaloids are significant  for  the  protection of plants  because  they ensure their  survival against  micro- organisms (antibacterial and antifungal activities) and act as feeding deterrents to insects and herbivores (Molyneux et al., 1996). The use of alkaloid-containing plants as dyes, spices, drugs or poisons can be traced back almost to the beginning of civilization. Alkaloids have many pharmacological activities including antihypertensive effects (many indole alkaloids), antiarrhythmic  effect  (quinidine,  sardine),  antimalarial  activity  (quinine),  and  anticancer actions (dimeric indoles, vincristine and vinblastine) (Wink et al., 1998). Some alkaloids have stimulant properties for example, caffeine and nicotine; morphine is used as analgesic and quinine as antimalarial drug (Rao et al., 1978).

1.4 Haematological Components and Their Functions

Blood is a connective tissue contained within a closed system of tubes (veins, arteries and capillaries) through which it is circulated by the pumping of the heart. Blood makes up 6-10% of the body mass in animals. It  is an unusual connective tissue that has specialized cells surrounded by blood cells themselves. The fluid matrix of the blood called plasma, makes up

55% of the blood volume, and surrounds the erythocytes (red blood cells) and cell fragments called platelet (thrombocytes) that make up 45% of the blood volume. Human blood is made up of the red blood cells, white blood cells, platelets and plasma in which they are suspended (Isaac et al., 2013).

1.4.1 Red blood cells and haemoglobin

Red blood cells make up almost 45 percent of the blood volume. Their primary function is to carry oxygen  from the  lungs  to  every  cell  in  the  body.  Red  blood  cells  are  composed predominantly of a protein and iron compound, called haemoglobin, which captures oxygen molecules as the blood moves through the lungs, giving blood its red colour. As blood passes through body tissues, haemoglobin then releases the oxygen to cells throughout the body. It is this haemoglobin that reacts with oxygen carried in the blood to form oxyhaemoglobin during respiration (Chineke et al., 2006). According to Isaac et al.(2013), red blood cell is involved in the transport of both oxygen and carbon dioxide in the body. Thus, a reduced red blood cell count implies a reduction in the level of oxygen that would be carried to the tissues as well as the level of carbon dioxide returned to the lungs (Ugwuene, 2011). Red blood cell count measures the number of red cells in the blood. A low count often accompanies anaemia, excess body fluid and blood loss. A high count is commonly seen in dehydration and in a condition called polycythaemia (Cheesbrough, 2006).

Haemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates with the exception of the fish family, channichthyldae (Sidell and O’ Brien,

2006) as well as tissues of invertebrates. Haemoglobin has the physiological function of transporting oxygen to tissues of the animal for oxidation of ingested food so as to release energy for the other body functions as well as transport carbon dioxide (Ugwuene, 2011; Isaac et al., 2013). Estimation of haemoglobin concentration is primarily used to determine the presence of anaemia or, its reverse, polycythaemia (Soetan et al., 2013).

1.4.2 White blood cells

White blood cells (WBCs), also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. The number of leukocytes in the blood is often an indicator of disease, and thus the WBC count is an important subset of the complete blood count (Cheesbrough, 2006). Animals with low white blood cells are exposed to high risk of disease infection, while those with high counts are capable of generating antibodies in the process of phagocytosis and thus, have high degree of resistance to  diseases (Soetan et  al., 2013)  and  enhanced adaptability to  local

environmental and disease prevalent conditions (Isaac et al., 2013). High counts are usually seen during infection, after exercise and with stress while abnormally low counts may be seen if there is suppression of the immune system. There are several types of white blood cells, including  neutrophils,  monocytes,  eosinophils,  basophils  and  lymphocytes,  all  of  which interact with one another and with plasma proteins and other cell types to form the complex and highly effective immune system (Cheesbrough, 2006).

1.4.3 Blood platelets

The smallest cells in  the  blood  are  the  platelets,  also  called  thrombocytes,  and  they  are designed for a single purpose; to begin the process of coagulation, or forming a clot, whenever a blood vessel is broken. As soon as an artery or vein is injured, the platelets in the area of the injury begin to clump together and stick to the edges of the cut. Low platelet concentration implies that  the  process of clot-formation (blood clotting)  will  be  prolonged resulting  in excessive loss of blood in the case of injury. Platelet count measures the number of platelets in blood. High platelet counts can be seen following strenuous activity, in some infections and inflammatory conditions. Extremely low platelet counts can be associated with spontaneous bleeding (Carlson, 1996).

1.4.4 Packed Cell Volume

Packed cell volume (PCV) which is also known as haematocrit (Ht or Hct) or erythrocyte volume fraction (EVF) is the percentage of red blood cells in blood (Carlson, 1996). It is the compact volume occupied by the red blood cells in a given volume of blood, expressed as a percentage. According to Isaac et al. (2013), the packed cell volume shows the capacity of blood to transport oxygen and nutrients. Thus an increased packed cell volume indicates a better transportation capacity of the red blood cells.

1.5 Antioxidants

Antioxidants are compounds that inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing-chain reactions, preventing further attack on other molecules (Eqbal et al., 2011). Antioxidants therefore serve to stabilize the highly reactive free radicals and reactive oxygen species, thereby maintaining the structural and functional integrity

of cells (Heo et al., 2004) and hence are very important to immune defence and health of humans and animals (Halliwell, 1995). Oxidative stress occurs when this critical balance is disrupted due to depletion of antioxidants or excess accumulation of reactive oxygen species, or  both  (Scandalios,  2005).  The  antioxidant  system  in  humans  involves  a  variety  of components, both endogenous and exogenous in origin, that function interactively and synergistically to neutralise free radicals (Jacob, 1995). These components include:

Exogenous antioxidants which include:

(a) Nutrient-derived antioxidants like ascorbic acid (vitamin C), tocopherols and tocotrienols (vitamin E), carotenoids, and other low molecular-weight compounds such as glutathione and lipoic acid (Sies and Stahl, 1995).

(b)  Numerous  other  antioxidant  phytonutrients  present  in  a  wide  variety  of  plant  foods

(Kuhnau, 1976).

Endogenous antioxidants which include the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, which catalyse free radical quenching reactions (Mates et al., 1999).

1.5.1 Exogenous antioxidants

1.5.1.1 Dietary antioxidants

Vitamin C and E and beta carotene (precursor of vitamin A) are among the most widely studied dietary antioxidants. 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 E, a major lipid-soluble antioxidant, is the most effective chain-breaking antioxidant within the cell membrane that protects membrane fatty acids from lipid peroxidation (Sies, 1992). Beta carotene and other carotenoids are also believed to provide antioxidant protection to lipid-rich tissues. Research suggests that beta carotene may work synergistically with vitamin E. A diet that is excessively low in fat may negatively affect beta carotene and vitamin E absorption, as well as other fat-soluble nutrients (Jacob, 1995; Sies and Stahl, 1995). Fruits and vegetables are major sources of vitamin  C  and  carotenoids,  while  whole  grains  and  high  quality,  properly extracted  and protected vegetable oils are major sources of vitamin E (Sies and Stahl, 1995).

1.5.1.2 Phytonutrients

A  number of other  dietary antioxidants exist  beyond  the  traditional vitamins  collectively known as phytonutrients or phytochemicals which are being increasingly appreciated for their antioxidant activity (Singh et al., 2009). Phytochemicals are bioactive non-nutrient chemical compounds found in plants that work with nutrients and dietary fibre to protect against diseases (Mohammed, 2013).They are secondary metabolites that contribute to flavor and color. Many phytochemicals have antioxidant activity and reduce the risk of many diseases (Agbafor and Nwachukwu, 2011). Their functions and mechanism of actions may include the following among others: antioxidant activity, hormonal action, stimulation of enzymes, interference with DNA replication and antibacterial properties (Sule and Mohammed, 2009).

1.5.2 Endogenous antioxidants

In addition to dietary antioxidants, the body relies on several endogenous defence mechanisms to help protect against free radical-induced cell damage. The antioxidant enzymes – glutathione peroxidase, catalase, and superoxide dismutase (SOD) – metabolise oxidative toxic intermediates and require micronutrient cofactors such as selenium, iron, copper, zinc, and manganese for optimum catalytic activity. It has been suggested that an inadequate dietary intake of these trace minerals may compromise the effectiveness of these antioxidant defense mechanisms (Sun et al., 1995; Hamid et al., 2010).

1.6 Antioxidant enzymes

The  utilization  of oxygen  for  metabolism  in  animals,  especially  humans,  has  led  to  the evolution of biochemical adaptations that exploit the reactivity of reactive oxygen species (ROS)  such as  superoxide (.O2–)  and  hydroxyl  radicals  (.OH-),  H2O2   and  singlet  oxygen (Graham and Christine, 1998). The most important antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and they convert reactive oxygen species into nonreactive oxygen molecules (Lee et al., 2004; Jinghua et al., 2007). They are an important protective mechanism against the reactive oxygen species and their effectiveness varies with the stage of development and other physiological aspects of the organism. These enzymes constitute a mutually supportive team of defense against ROS.

While superoxide dismutase lowers the steady-state level of O2–, catalase and peroxidases do the same for H2O2 (Jinghua et al., 2007).

1.6.1 Superoxide dismutase

Superoxide radical (.O2–) is generated as by-product in aerobic organisms from a number of physiological reactions and redox reactions in cells (Andrea et al., 1989). It can react with hydrogen peroxide  (H2O2)  to  produce  hydroxyl  radical  (.OH-),  one  of the  most  reactive molecules in the living cells. Hydroxyl radical can cause the peroxidation of membrane lipids. To  ameliorate  the  damage  caused  by  hydroxyl  radical,  superoxide radical  and  hydrogen peroxide, organisms have evolved mechanisms to control the concentration of these oxidants (Campana et al., 2004). Superoxide dismutase (SOD) is a well-known antioxidant enzyme that converts superoxide into hydrogen peroxide (Henry et al., 2006). In humans, there are three forms of SOD: cytosolic Cu/Zn-SOD, mitochondrial Mn-SOD, and extracellular SOD (EC- SOD) (Sandstrom et al., 1994; Mates et al., 1999). Superoxide dismutase destroys O2-.by successive oxidation and reduction of the transition metal ion at the active site in a ping pong type mechanism with remarkably high reaction rates (Turrens et al., 1984). The enzyme mainly acts by quenching of superoxide radicals in a reaction where it spontaneously dismutates O .–

anion to form O2 and H2O2. (Vivek and Surendra, 2006).

.

O2– +  O2.–  + 2H+       superoxide dismutase       H2O2   +  O2                 (1)

1.6.2 Catalase

Catalase (EC 1.11.1.6) (H2O2 oxidoreductase) is an enzyme antioxidant widely distributed in all animal tissues. It catalyses the decomposition of H2O2  to oxygen and water in order to neutralize the effects of hydrogen peroxide generated within the cells (Noreddine et al., 2005). Catalase is one of the most efficient enzymes known and it has one of the highest turnover rates of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. Decomposition of H2O2 by the catalytic activity of catalase follows the fashion of a first-order reaction and its rate is dependent on the concentration of H2O2 (Kabel, 2014).

2H2O2        catalase                                  2H2O + O2                                                               (2)

1.6.3 Glutathione peroxidase

Glutathione peroxidase (GPx)  is  an enzyme  that  is  responsible  for  protecting  cells  from damage due to free radicals like hydrogen peroxide and lipid peroxides. The GPx contains a single selenocysteine (Sec) residue in each of the four identical subunits, which is essential for enzyme activity (Speranza et al., 1993). Glutathione peroxidases (EC 1.11.1.19) are important for extra-peroxisomal inactivation of hydrogen peroxide generated by superoxide dismutase (Glantzounis et al., 2005). These selenium-containing enzymes use glutathione (GSH) as a substrate when converting hydrogen peroxide to water. In this process the cysteine thiol group of GSH is oxidized to GS· radical, which is further dismutated to glutathione disulphide (GSSG). Glutathione reductase (GR) regenerates GSH from GSSG in a reaction that requires NADPH.

ROOH + 2GSH    glutathione peroxidase              ROH + GSSG + H2O                    (3)

1.7 Oxidative stress

The ability of the cell to utilize oxygen has provided humans with the benefit of metabolizing fats, proteins, and carbohydrates for energy; however, it does not come without cost (Halliwell and Gutteridge, 1998). Oxidative stress occurs when the production of reactive oxygen metabolites exceeds the capacity of the antioxidant system of the cell, tissue or body (Moussa,

2008), hence, the shift in the balance between oxidants and antioxidants in favor of oxidants is termed “oxidative stress” (Birben et al., 2012). It  is a deleterious process that can be an important mediator of damage to cellular structures such as lipids and membranes, proteins, and DNA (Valko et al., 2007). If unchecked, these lead to diseases of many organs such as cancer, atherosclerosis, ageing, liver damage, diabetes and Parkinson’s disease (Niture et al.,

2014). Oxidative stress is believed to aggravate the symptoms of many diseases, including haemolytic  anaemias,  beta-haemoglobinopathies  (sickle  cell  anaemia  and  thalassemia), glucose-6-phosphate dehydrogenase deficiency,  congenital dyserythropoietic anaemias  and paroxysmal nocturnal haemoglobinuria (Fibach and Rachmilwitz, 2008). Although oxidative

stress is not the primary aetiology of these diseases, oxidative damage to their erythroid cells plays a crucial role in haemolysis due to ineffective erythropoiesis in the bone marrow and short survival of red blood cells  in the circulation (Aster, 2004). Moreover, platelets and polymorphonuclear (PMN)  white  cells  are  also  exposed  to  oxidative  stress  (Fibach  and Rachmilwitz, 2008).

1.7.1 Lipid peroxidation

The erythrocyte membrane is an integrated system of lipids, proteins and carbohydrates. It has two  important  properties. First,  the  molecular  properties of lipids  and  proteins  and  their supramolecular organization  are  important  in  membrane  structure  and  deformability,  and secondly in their function, relating to enzyme activities, ionic pump channels, transports and receptor activities (Edwards and Fuller, 1996). In addition to containing high concentrations of polyunsaturated fatty acids and transition metals, red blood cells are constantly being subjected to various types of oxidative stress. The red blood cell however, represents an important component of the antioxidant capacity of blood, comprising in particular, intracellular enzymes like superoxide dismutase, catalase and also the glutathione system (Grune et al., 2000). Lipid peroxidation is a complex process whereby polyunsaturated fatty acids (PUFAs) in the phospholipids of cellular membranes undergo reaction with oxygen to yield lipid hydroperoxides (LOOH). The reaction occurs through a free radical chain mechanism initiated by the abstraction of a hydrogen atom from a PUFA by a reactive free radical, followed by a complex sequence of propagative reactions (Palmieri and Sblendorio, 2007). This leaves an unpaired electron on the carbon, forming a carbon-centered radical, which is stabilized by a molecular rearrangement of the double bonds to form a conjugated diene which then combines with oxygen to form a peroxyl radical. The peroxyl radical is itself capable of abstracting a hydrogen atom from another polyunsaturated fatty acid and so of starting a chain reaction (Repetto  et  al.,  2012).  The  inhibition of enzymatic  lipid  oxidation  may  be  achieved  by inhibition  of  either  the  activation  or  reaction  of an  enzyme.  Free  radical-mediated  lipid peroxidation may be inhibited by the inhibition of chain initiation and chain propagation and/or acceleration  of chain  termination.  Lipid  peroxidation induced  by  singlet  oxygen  may  be inhibited by the inhibition of its formation by, for example, quenching of ultraviolet light and quenching of the singlet oxygen itself by carotenoids (Niki et al., 2005). Therefore, toxicants

with ability of inducing lipid peroxidation can cause great harm on tissues rich in unsaturated fatty acid, such as the brain. The final metabolites from lipid peroxidation are also reactive and can damage cells further (Boelsterli, 2009).

Molecular oxygen rapidly adds to the carbon-centered radicals (R.) formed in this process, yielding  lipid  peroxyl  radicals  (ROO.).  The  formation  of  peroxyl  radicals  leads  to  the production of organic hydroperoxides, which in turn, can abstract hydrogen from another

PUFA.

Figure 2: Initial phase of the propagation step of lipid peroxidation process indicating the oxygen uptake.

Source: Halliwell, and Gutteridge, 1984.

Lipid peroxidation causes a decrease in membrane fluidity and in the barrier functions of the membranes. The toxicity of lipid peroxidation products in mammals generally involves neurotoxicity, hepatotoxicity and nephrotoxicity (Boveris et al., 2008).

Lipid peroxidation or reaction of oxygen with unsaturated lipids produces a wide variety of oxidation products. The main primary products of lipid peroxidation are lipid hydroperoxides (LOOH). Among the many different aldehydes which can be formed as secondary products during lipid peroxidation, malondialdehyde (MDA), propanal, hexanal, and 4-hydroxynonenal (4-HNE) (Esterbauer et al., 1991), MDA appears to be the most mutagenic product of lipid peroxidation, whereas 4-HNE is the most toxic (Esterbauer et al., 1990).

1.7.2 Malondialdehyde

Malondialdehyde (MDA) has been widely used for many years as a convenient biomarker for lipid peroxidation of omega-3 and omega-6 fatty acids because of its facile reaction with thiobarbituric acid (TBA) (Pryor, 1989). The TBA test is predicated upon the reactivity of TBA toward MDA to yield an intensely colored chromogen fluorescent red adduct; this test was first used by food chemists to evaluate autoxidative degradation of fats and oils (Sinnhuber et al., 1958). Malondialdehyde is one of the most popular and reliable markers that determine oxidative stress in clinical situations (Schauenstein, 1967). Malondialdehyde is believed to originate under stress conditions and has high capability of reaction with multiple biomolecules such as proteins or DNA that leads to the formation of adducts (Blair, 2008; Zarkovic et al.,

2013), and excessive MDA production have been associated with different pathological states

(Blair, 2008).

Once formed, MDA can be enzymatically metabolized or can react with cellular and tissular proteins or DNA to form adducts resulting in biomolecular damages. Initial reactions between MDA and free amino acids or protein generate Schiff-base adducts (Del Rio et al., 2005). These  adducts  are  also  referred  to  as  advanced  lipid  peroxidation end-products (ALEs). Acetaldehyde (product of MDA metabolism) under oxidative stress and in the presence of MDA further generates malondialdehyde acetaldehyde (MAA) adducts (Blair, 2008). MDA adducts are biologically important because they can participate in secondary deleterious reactions (e.g.,  cross-linking)  by promoting  intramolecular or  intermolecular protein/DNA cross-linking   that   may   induce   profound   alteration   in   the   biochemical  properties   of biomolecules and accumulate during ageing and in chronic diseases (Negre-Salvayre et al.,

2008). MDA is an important contributor to DNA damage and mutation and in the absence of

repair, MDA-DNA adducts may lead to mutations (point and frameshift) (Niedernhofer, 2003), strand breaks, cell cycle arrest, and induction of apoptosis (Ji et al., 1998; Willis et al., 2004).

1.8 Justification for the study

The present investigation is a continuation of the research efforts aimed at  providing the requisite  scientific  information and  empirical data on the  therapeutic  value,  toxicological potential and active principles of Nigerian medicinal plants as used in the treatment  and management of several ailments. Jatropha tanjorensis has recently received a lot of attention for both its nutritional and antioxidant properties and several researchers have shown the leaves to have hypoglycemic (Ijioma et al., 2014), hypolipodermic (Oyewole and Akingbala, 2011), antioxidant (Omoregie et al., 2011), antimicrobial (Oboh and Masodje, 2009) and anti-anaemic (Idu et al., 2014) properties among others. The present study is designed to validate the ethnomedicinal use of Jatropha tanjorensis using methanol as the solvent for extraction. Thus, the study is out to determine the veracity of the claims to the antioxidant effect and improved haematological state of animals treated with Jatropha tanjorensis leaf extracts. This will also serve as criteria to recommend for the continuous use of the plant in ethnopharmacology.

1.9 Aim of the study

The aim of this study was to investigate the effects of oral administration of the methanol extract of J. tanjorensis on some haematological parameters and antioxidant enzymes in albino rats.

1.10 Specific objectives of the research

(i)  To  determine  qualitatively  and  quantitatively,  the  phytochemical  constituents  of  the methanol extract of J. tanjorensis

(ii) To determine the median lethal dose (LD50) of the methanol extract

(iii) To assess the effects of the methanol extract treatment on some haematological indices

(iv) To  assess the  effects of the  methanol extract  treatment  on Malondialdehyde (MDA)

concentration. (v)  To  assay  the  activity  of  each  of  superoxide  dismutase  (SOD),  catalase  (CAT)  and glutathione peroxidase (GPx).

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