THE IN VIVO AND IN VITRO ANTIOXIDANT EFFECT OF THE FLAVONOID- RICH FRACTION OF THE METHANOL EXTRACT OF JATROPHA TANJORENSIS LEAVES

ABSTRACT

The leaves of Jatropha tanjorensis are edible and used in herbal medicine in the treatment of diseases associated with oxidative stress.  The  pharmacological value  of the  plant  is  partly associated with its phytochemical components which produce physiological actions in the human body. In the present study, the qualitative and quantitative phytochemical analyses of the crude extract of J. tanjorensis leaves showed the presence of alkaloids (7.00%), flavonoids (20.00%) saponins (2.00%) and tannins (2.30%) while steroids were not detected. The total flavonoid concentration (TFC) of the ethylacetate fraction was 382.60±1.74 mg QE/g. The flavonoid-rich fraction of J.  tanjorensis (FRJT) at  various concentrations (0.125 – 1.00  mg/ml) exhibited concentration-dependent increase in total antioxidant capacity (TAC). In the FRAP assay, the reduction of Fe3+ to Fe2+ at different concentrations (15.70-1000 µg/ml) of FRJT was also dose- dependent. There was relative increase in the inhibition (%) of DPPH radicals with increased concentrations (15.70-1000 µg/ml) of FRJT, while the EC50 was 469 µg/ml. The acute toxicity test showed no mortality up to 5 ml/kg (5000 mg/kg) body weight, which indicated the possible safety of FRJT. Assay of antioxidant enzymes (SOD, CAT and GPx) showed that the activities of CAT and GPx were significantly (p ˂ 0.05) lower while that of SOD was found to be non- significantly (p ˃ 0.05) lower in the untreated animals (group 2) when compared to the normal control (group 1). In contrast, SOD activity was found to be significantly (p ˂ 0.05) higher in groups 4 and 5. CAT activity was non-significantly (p ˃ 0.05) higher in groups 3 and 4 whereas GPx activity was found to be significantly (p ˂ 0.05) higher in groups 3 and 4 when compared to the untreated group. There was significant (p < 0.05) elevation in the activities of liver marker enzymes such as AST, ALT and  ALP of untreated animals when compared to the normal control. However a significant reduction (p < 0.05) was observed across all the pre-treated groups (Groups 3 – 5) when compared to the untreated group. Serum concentrations of urea and creatinine were found to be significantly (p < 0.05) higher as observed in the untreated group when compared to group 1. Conversely, the concentration of creatinine was significantly (p < 0.05) lower in group 4 and non-significantly (p > 0.05) lower in groups 3 and 5 when compared to the untreated group. The concentration of urea was found to be non-significantly (p < 0.05) lower across all the pre-treated animals (Groups 3 – 5) when compared to the untreated group. PCV, Hb and WBC levels were significantly (p < 0.05) lower in group 2 when compared to group 1. However, supplementation with FRJT and Silymarin ameliorated the induced depletion of blood in the pre-treated animals (Groups 3 – 5). Histological examination of the liver tissue showed marked reduction in fatty degeneration across the pre-treated groups when compared to the untreated group. These results indicate that the flavonoid-rich fraction of J. tanjorensis was able to establish antioxidant effect on in-vitro models. FRJT also exhibited hepatoprotective properties by restoring liver enzymes during CCl4-induced oxidative stress in rat models. These results also indicate that the flavonoid-rich fraction contains antioxidants, which mop up free radicals in the system and support its use in the treatment of diseases resulting from oxidative damage.

CHAPTER ONE

INTRODUCTION

Primary healthcare  systems  involve  use  of  medicinal plants as  an effective  source of  both traditional and modern medicines. Any plant which possesses curative elements or properties in one or more of its parts may be termed a medicinal plant. Plant based medications have been employed since the dawn of civilization for prolonging the life of man and for combating various ailments. World Health Organization (WHO) also advocates use of traditional medicines as safe remedies for ailments of both microbial and non-microbial origins (Goel and Sharma, 2014). According to World Health Organization, more than one billion people rely on herbal medicines to some extent. Goel and Sharma (2014) also reported that 21,000 plants all around the world have been listed for their medicinal uses and it has been estimated that as many as 80 percent of the world’s population depends on these plants for their primary healthcare needs. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases. Medicinal value of plants depends on these inherent substances that produce a definite physiological action on the human body. In developing countries herbal medicine still serves as the mainstay of about 75-80% of the whole population (Parekh et al., 2005). Herbal medicine has better cultural acceptability, better compatibility with the human body and fewer side effects (Goel and Sharma, 2014). Plants have provided a source of inspiration for novel drug compounds, as plant derived medicines have made large contributions to human health and well- being (Avijgan et al., 2010). Early humans recognized their dependence in nature in both health and illness. Led by instinct, test and experience, primitive people treated illness by using plant, animal parts and minerals that were not part of their diet (Anwanni and Alta, 2005).  In recent years, there has been a gradual revival of interest in the use of medicinal plants in developing countries because herbal medicines have been reported to be well tolerated when compared with synthetic drugs (Iniaghe et al., 2009).

Nigeria is considered to have a rich reservoir of medicinal plants with potential chemotherapeutic constituents. Traditional healers in Nigeria play a major role as health care providers in both urban and rural settings. These traditional medical practitioners are knowledgeable regarding the use of plants and plant extracts for healing purposes. This combination is valuable in the search for new drug remedies based on ethnomedical information (Avijgan et al., 2009).

1.1 Review on Jatropha tanjorensis

Jatropha tanjorensis is a perennial herb, a member of the Euphorbiaceae family, commonly called “hospital too far” or “Catholic vegetable” in southern Nigeria (Omoregie and Osagie,

2012). It shows intermediacy in phenotypic characters between Jatropha curcas and Jatropha gossypifolia (Prabakaran and Sujatha, 1999). Jatropha tanjorensis is a native of Central America and has become naturalized in many tropical and subtropical countries, including Africa, India and North America (Prabakaran and Sujatha, 1999). According to Oboh and Masodje (2009), Jatropha tanjorensis is  predominantly grown in southern Nigeria and  is  primarily used  for fencing. The leaves of the plant are a source of edible leafy vegetable and taken as a tonic in herbal medicine, with the claim that it increases blood volume (Omoregie and Sisodia, 2011). Traditionally, decoction of the leaves is used to treat anaemia (as a haematinic agent), diabetes, skin diseases, malaria, and cardiovascular diseases (Iwalewa et al., 2005; Oduola et al., 2005). However, the plant’s popularity was doused by unproven claims that the whitish latex emanating from the leaf stem and stalk (which causes irritation and mild rashes) may be toxic to man (Omoregie and Sisodia, 2011). Jatropha tanjorensis has received a lot of attention due to its potential health benefits, availability and affordability.

Phytochemical analysis of the leaves showed the presence of flavonoids, tannins, terpenoids, saponins and cardiac glycosides (Oyewole and Akingbala, 2011). They further stated that these phytochemical ingredients have hypolipidemic properties in the blood of rats and concluded that the medicinal properties attributed to J. tanjorensis as a useful herb in the treatment of heart diseases could be based on the antioxidant properties and positive modulatory effects of its phytochemicals on serum lipid profile in albino rats. Reports also showed that J. tanjorensis is rich in antioxidant nutrients like phosphorus, selenium, zinc and vitamins C and E (Omobuwanjo et al., 2011). Iwalewa et al. (2005) reported that J. tanjorensis exhibited low antioxidant and very low hemaglutination titre value, the later indicating low toxicity on red blood cells. Reports on in vitro and in vivo antioxidant properties of methanolic extracts of J. tanjorensis on nutritionally stressed rats (protein malnutrition) confirmed the local claims on the efficacy of the plant leaves, stating that it may provide effective intervention for free radical mediated diseases (Omoregie and Osagie, 2012). Furthermore, Atansuyi et al. (2012) also studied the in vitro antioxidant properties of free and bound phenolic extracts of the leaves of J. tanjorensis and the extracts inhibited Fe2+-

induced hepatic and cerebral lipid peroxidation process. Antimicrobial studies of J. tanjorensis showed that the aqueous extract of the leaves inhibited gram +ve bacterium Staphylococcus aureus and gram –ve bacterium Escherichia coli (Oboh and Masodge, 2009). Crude ethanolic extracts of the plant leaves exhibited relatively high antiplasmodial and low cytotoxic activities; this  may  be  attributed to  the  presence of some  inherent  phytochemicals which  might  have conferred some protective / antioxidative effect against oxidative stress induced by the malaria parasite  (Omoregie and  Sisodia,  2011).  Studies  on the  aqueous  extract  of the  leaves  of J. tanjorensis showed a statistically significant increase on the PCV and Heamoglobin concentration of both male and female wistar rats, thereby justifying the local claim of the plant’s use as a blood tonic (Omigie et al., 2013).

Finally, toxicity and histopathological studies of the leaf extract on rats revealed no significant abnormalities in the tissues except for mild effects on the lungs and liver (Omobuwanjo, 2011). The wide usage of the leaves of J. tanjorensis for treatment of diseases in folklore medicine can be attributed to its high polyphenolic content. Hence, it could be speculated that the plant utilizes several antioxidant mechanisms to elicit its pharmacological effect.

1.2 Scientific classification of Jatropha tanjorensis

Jatropha tanjorensis is taxonomically classified as follows: Kingdom                    Plantae (plants)

Subkingdom              Tracheophyta (vascular plants) Division                     Magnoliophyta (flowering plants) Class                           Magnoliopsida (dicotyledons) Order                          Malpighiales

Family                        Euphorbiaceae

Genus                         Jatropha

Species                       Jatropha tanjorensis

Source: (Species 2000 & ITIS Catalogue of Life, 2013)

Fig 1: Jatropha tanjorensis leaves

1.3 Review on phytochemistry

Phytochemicals are  non-nutritive plant  chemicals  that  have  protective or  disease  preventive properties (Ene et al., 2013). They are non-essential nutrients meaning that they are not required by the body for sustaining life. Ene et al. (2013) reported that plants produce these chemicals to protect themselves, but recent researches demonstrate that they can also protect humans against diseases. These chemicals are often referred to as “secondary metabolities”. There are more than one thousand known phytochemicals. They include alkaloids, flavonoids, coumarins, saponins, glycosides, gums, polysaccharides, phenols, tannins, terpenes and terpenoids (Harborne, 1998; Okwu, 2005). The most important of these phytochemicals are alkaloids, tannins, flavonoids and phenolic compounds (Iwu, 2000).

Different mechanisms have been suggested for the action of phytochemicals. They may act as antioxidants, or modulate gene expression and signal transduction pathways ((Dandjesso et al,

2012). They may be used as chemotherapeutic or chemopreventive agents (Paolo et al., 1991). The subject of phytochemistry to plant chemistry has developed in recent years as a distinct discipline, somewhere in between natural product, organic chemistry and plant biochemistry and

is closely related to both. It is concerned with the enormous varieties of organic substances that are elaborated and accumulated by plant and deals with chemical structure of these substances, their biosynthesis, turnover and metabolism, their natural distribution and biological functions (Bouchet et al., 1982).

The main aim of phytochemical screening is to identify the nature of the compounds present in a given plant extract, which may be responsible for the observed biological effect. Medicinal action of some species of plant is as a result of the effect of the plant constituents on some of the organs of the human body. They clear up residual symptoms or destroy the cause of the disease, in most cases infectious microorganisms. They increase the body’s resistance to disease, retard or ease the process of natural ageing. These components are responsible for a green therapeutic effect and they frequently serve as model for the synthesis of new medicine (David et al., 1997).

1.3.1   Flavonoids

Flavonoids (or bioflavonoids) are water soluble polyphenolic molecules containing 15 carbon atoms. (Batra and Sharma, 2013). Flavonoids were referred to as vitamin P (probably because of the effect they had on the permeability of vascular capillaries) from the mid-1930s to early 50s, but  the  term has since  fallen out of use  (Mobh, 1938).  In Fig.2  below,  flavonoids can be visualized as two benzene rings which are joined together with a short three carbon chain. One of the carbons of the short chain is always connected to a carbon of one of the benzene rings, either directly or through an oxygen bridge, thereby forming a third middle ring, which can be five or six-membered.  Flavonoids  consist  of  6   major  subgroups:  chalcone,  flavones,  flavonols, flavanones, anthocyanins and isoflavonoids (Robak and Gryglewski, 1988). Together with carotenes, these flavonoids are also responsible for the coloring of fruits, vegetables and herbs (Harnly et al., 2006). They are found in most plant materials and the most important dietary sources are fruits, tea and soybean while green and black tea contains about 25% flavonoids (Alexopoulos et al., 2008). Other important sources of flavonoids are apple (quercetin), citrus fruits (rutin and hesperidin), watermelons, berries, onions, tomatoes, vegetables and herbs (Harnly et al., 2006).

Fig 2: Molecular structure of the flavonoid backbone and major subgroups. Source: (Batra and Sharma, 2013)

1.3.1.1 Health benefits of flavonoids

Flavonoids have antioxidant activity and have become very popular because they have many health promoting effects. Some of the activities attributed to flavonoids include: antioxidant, anti- allergic, anti-cancer, anti-inflammatory, anti-microbial, anti-diarrheal activities as well as nervous and cardiovascular system benefits (Kozlowska and Szostak-Wegierek, 2014). The flavonoid quercetin is known for its ability to relieve hay fever, eczema, sinusitis and asthma. Epidemiological studies have illustrated that heart diseases are inversely related to flavonoid intake and several studies have also shown that flavonoids prevent the oxidation of low-density lipoprotein thereby reducing the risk for the development of atherosclerosis (Cermak, 2008). The contribution of flavonoids to the total antioxidant activity of components in food can be very high because daily intake can vary between 50 to 500 mg (Chun et al., 2007). Red wine contains high levels of flavonoids, mainly quercetin and rutin. The high intake of red wine (and flavonoids) by the French might explain why they suffer less from coronary heart disease than other Europeans, although their consumption of cholesterol rich foods is higher (French paradox). Many studies have confirmed that one or two glasses of red wine daily can protect against heart disease. Tea flavonoids reduce the oxidation of low-density lipoprotein, lowers the blood levels of cholesterol and triglycerides (Kozlowska and Szostak-Wegierek, 2014). Soy flavonoids (isoflavones) reduce blood cholesterol, prevent osteoporosis and used to ease menopausal symptoms (Azadbakht et al.,

2007).

Flavonoids have also been shown to inhibit topoisomerase enzymes (Esselen et al., 2009; Bandele et al., 2008) and to induce DNA mutations in the mixed-lineage leukemia (MLL) gene in in vitro studies (Barjesteh van Waalwijk et al., 2007). However, in most of the above cases no follow up in vivo or clinical research has been performed, leaving it impossible to say if these activities have any beneficial or detrimental effect on human health (Esselen et al., 2009).

1.3.1.2 Role of Flavonoids as antioxidant

Many flavonoids (especially those belonging to two flavonoid subgroups called flavonols and flavan-3-ols) can be effective in reducing free radical damage to cells and other components in body tissue; they provide antioxidant benefits (Kozlowska and Szostak-Wegierek, 2014). It is not clear,  however,  if  flavonoids  fall  in  the  same  category as  more  widely  known antioxidant nutrients like vitamin C or vitamin E. The reason for this is because their concentration in the bloodstream is so  much lower. Another reason lies in the fact that many of the antioxidant functions of flavonoids are not performed by the flavonoids themselves, but by forms of the flavonoids that have been altered by our metabolism. The way flavonoids function as antioxidants is not well known in detail , however, studies have documented better protection of certain cell types (for example, red blood cells) following consumption of flavonoid-rich foods. Blueberries, for example, have been repeatedly studied in this context for their flavonoid-related antioxidant benefits (Kozlowska and Szostak-Wegierek, 2014).

In this antioxidant context, it  is also  worth pointing out, the potentially unique relationship between flavonoids and vitamin C. Recent studies have shown the ability of flavonoids to alter transport of vitamin C, as well as to alter function of an enzyme called ascorbate oxidase, which converts vitamin C into a non-vitamin form (monodehydroascorbate). Therefore, the transport and cycling of vitamin C is flavonoid related. This association makes sense, since so many foods high in vitamin C (such as citrus fruits, papaya, bell peppers, broccoli, Brussels sprouts, and strawberries) are also high in flavonoids (Harnly et al., 2006).

Research at the Linus Pauling Institute and the European Food Safety Authority have shown that flavonoids are poorly absorbed in the human body (less than 5%), with most of what is absorbed being quickly metabolized and excreted (Lotito and Frei, 2006; Williams et al., 2004). These findings  suggest  that  flavonoids  have  negligible  systemic  antioxidant  activity,  and  that  the

increase in antioxidant capacity of blood seen after consumption of flavonoid-rich foods is not caused directly by flavonoids, but is due to production of uric acid resulting from flavonoid depolymerization and excretion (Lotito and Frei, 2006).

1.3.2 Alkaloids

Alkaloids are a group of naturally occurring chemical compounds that  contain mostly basic nitrogen atoms (Ene et al., 2013). This group also includes related compounds (with natural or even weakly acidic properties). Some synthetic compounds of similar structure are also attributed to alkaloids. In addition to carbon, hydrogen and nitrogen, alkaloids also contain oxygen, sulphur and more rarely other elements such as chlorine, bromine and phosphorus (Ene et al., 2013). Alkaloids have an array of structural types, biosynthetic pathways, and pharmacological activities (Tanko et al., 2008). In plants and insects, toxic alkaloids are sequestered for use as a passive defense mechanism by acting as deterrents for predating insects (Eyong et al., 2006). Alkaloids have been used throughout history in folk medicine in different regions around the world. They have been a constituent part of plants used in phytotherapy. Many of the plants that contain alkaloids are just medicinal plants and have been used as herbs. Some alkaloids that have played an important role in this sense include aconitine, atropine, colchicine, coniine, ephedrine, ergotamine, mescaline, morphine, strychnine, psilocin and psilocybin (Aladesanmi et al., 1998). Many alkaloids are known to have effect on the central nervous system. Some alkaloids act as analgesic (such as morphine, a pain killer). Quinine was widely used against Plasmodium falciparum. In this respect, it is found from the phytochemical screening that most plants traditionally used to treat malaria contain alkaloids among other things (Tor-anyiin et al., 2003; Jeruto et al., 2011).

1.3.3 Saponins

Saponins are glycosides of both triterpenes and steroids that are characterized by their bitter or astringent taste, foaming property Okigbo et al., 2009), (haemolytic effect on red blood cells and cholesterol binding properties (Okwu, 2005). Saponins increase the permeability of intestinal mucosa cells, inhibit active nutrient transport, and facilitate the uptake of substances to which the gut  would  normally be  impermeable (Gee et  al., 1997). It  has also  been shown to  possess beneficial  effects  such  as  cholesterol  lowering  properties  and  exhibits  structure  dependent

biological activity (Harborne, 1998). Saponins, being both fat and water soluble, have surfactant and detergent activity. Thus they would be expected to influence emulsification of fat-soluble substances in the gut, including the formation of mixed micelles containing bile salts, fatty acids and fat-soluble vitamin (Okigbo et al., 2009).

1.3.4 Tannins

Tannins  are  an  exceptional  group  of  water  soluble  phenolic  metabolites  of  relatively  high molecular weight and having the ability to complex strongly with carbohydrates and proteins (Heldt and Heldt, 2005). Tannins are astringent, bitter plant polyphenols and the astringency from tannins is what causes the dry and pucker feeling in the mouth following the consumption of unripened fruit or red wine (Serafini et al., 1994). They are grouped into two forms hydrolysable and condensed tannins (Nityanand, 1997). Hydrolysable tannins are potentially toxic and cause poisoning if large amounts of tannin-containing plant material such as leaves of oak (Quercus spp.) and yellow wood (Terminalia oblongata) are consumed (Heldt and Heldt, 2005) and as such seen as one of the anti-nutrients of plant origin because of their capability to precipitate proteins, inhibit the digestive enzymes and decline the absorption of vitamins and minerals (Khattab et al.,

2010). 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 and lipoxygenases in vitro (Amarowicz et al., 2000). They have also been shown to possess antimicrobial, antiviral antimutagenic and antidiabetic properties (Gafner et al., 1997). The antioxidant activity of tannins results from their free radical and reactive oxygen species-scavenging properties, as well as the chelation of transition metal ions that modify the oxidation process (Serrano et al., 2009).

1.3.5  Steroids

Sterols are  triterpenes which  are  based on the  cyclopentane  hydrophenanthrene ring  system (Harborne, 1998). Sterols in plants are generally described as phytosterols with three known types occurring in higher plants: sitosterol (formerly known as β-sitosterol), stigmasterol and campsterol (Harborne, 1998). These common sterols occur both as free and as simple glucosides. Sterols have essential functions in all eukaryotes. Free sterols are integral components of the membrane lipid bilayer where they play important role in the regulation of membrane fluidity and permeability

(Irvine, 1961). While cholesterol is the major sterol in animals, a mixture of various sterols is present  in  higher plants,  with  sitosterol usually  predominating. However, certain  sterols  are confined to lower plants such as ergosterol found in yeast and many fungi while others like fucoterol, the main steroid of many brown algae is also detected in coconut (Harborne, 1998).

1.4 Oxidative challenge in biology

A paradox in metabolism is that, while the vast majority of complex life on earth requires oxygen for  its  existence,  oxygen  is  a  highly  reactive  molecule  that  damages  living  organisms  by producing reactive oxygen species (Davies, 1995). Consequently, organisms contain a complex network of antioxidant metabolites and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids (Sies, 1997; Vertuani et al., 2004). In general, antioxidant systems either prevent these reactive species from being formed, or remove them before they can damage vital components of the cell (Davies 1995; Sies, 1997). However, reactive oxygen species also have useful cellular functions, such as redox signaling. Thus, the function of antioxidant systems is not to remove oxidants entirely, but instead to keep them at an optimum level (Rhee, 2006).

2

The reactive oxygen species produced in cells include hydrogen peroxide (H2O2), hypochlorous acid (HClO), and free radicals such as the hydroxyl radical (·OH) and the superoxide anion (O- ) (Valko et al., 2007).The hydroxyl radical is particularly unstable and will react rapidly and non- specifically with most biological molecules. This species is produced from hydrogen peroxide in

metal-catalyzed redox reactions such as the Fenton reaction (Stohs and Bagchi, 1995). These oxidants can damage cells by starting chemical chain reactions such as lipid peroxidation, or by oxidizing DNA or proteins (Sies, 1997). Damage to DNA can cause mutations and possibly cancer, if not reversed by DNA repair mechanisms, while damage to proteins causes enzyme inhibition, denaturation and protein degradation (Stadtman, 1992). The use of oxygen as part of the process for generating metabolic energy produces reactive oxygen species (Raha and Robinson, 2000). In this process, the superoxide anion is produced as a by-product of several steps in the electron transport chain. Particularly important is the reduction of coenzyme Q in complex III, since a highly reactive free radical is formed as an intermediate (Q·−). This unstable intermediate can lead to electron “leakage”, when electrons jump directly to oxygen and form the superoxide anion, instead of moving through the normal series of well-controlled reactions of the

electron  transport  chain  (Finkel  and  Holbrook,  2000).  Peroxide  is  also  produced  from  the oxidation of reduced flavoproteins, such as complex I (Hirst et al., 2008). However, although these enzymes can produce oxidants, the relative importance of the electron transfer chain to other processes that generate peroxide is unclear (Seaver and Imlay, 2004;  Imlay, 2003). In plants, algae,  and  cyanobacteria,  reactive  oxygen species  are  also  produced  during  photosynthesis, particularly under conditions of high light intensity (Krieger-Liszkay, 2004). This effect is partly offset by the involvement of carotenoids in photoinhibition, and in algae and cyanobacteria, by large amount of iodide and selenium, which involves these antioxidants reacting with over- reduced forms of the photosynthetic reaction centres to prevent the production of reactive oxygen species (Szabó et al., 2005).

1.4.1 Oxidative stress

Oxidative stress is damage to cell structure and cell function by overly reactive oxygen-containing molecules and chronic excessive inflammation (mostly caused by xenobiotics or foreign substances) (Sies, 1997). Free radicals possess an unpaired electron in their outermost shell and are capable of independent existence (Halliwell and Gutterridge, 1999). Their half-lives vary from a few  nanoseconds for the most reactive compounds to seconds and  hours for rather stable radicals. They trigger chain reactions resulting in the oxidation of macromolecules in order to reach a steady state (Aristidis et al., 2012). Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells.

Reactive oxygen species and free radicals formed during oxidation have been reported to contribute to diseases such as cancer, diabetes and ageing (Halliwell and Gutterridge, 1999). Other  diseases caused  by oxidative stress  include Alzheimer’s and  Parkinson’s diseases,  the pathologies caused by diabetes, rheumatoid arthritis, and neurodegeneration in motor neuron diseases (Wood-Kaczmar et al., 2006). In many of these cases, it is unclear if oxidants trigger the disease, or if they are produced as a secondary consequence of the disease and from general tissue damage. One case in which this link is particularly well-understood is the role of oxidative stress in cardiovascular disease. Here, low density lipoprotein (LDL) oxidation appears to trigger the process of atherogenesis, which results in atherosclerosis, and finally cardiovascular disease. A low calorie diet extends median and maximum lifespan in many animals. This effect may involve a reduction in oxidative stress (Sies, 1997).

Oxidative stress  interferes with  many cellular  functions, such as cell  cycle  progression and apoptotic  pathways  that  can  reduce  the  ability  of antineoplastic  agents  to  kill  cancer  cells (Conklin, 2004). The effects are mediated, most likely, by the many aldehydes that result from oxidative stress-induced lipid peroxidation. Conklin (2004) stated that since many drugs used for cancer chemotherapy cause oxidative stress (which can interfere with antineoplastic activity), reducing this oxidative stress by administering antioxidants may enhance the effectiveness of the treatment; However, enhancing the cytotoxicity of antineoplastic agents would affect normal cells as well as cancer cells.

1.4.2 Mediators of Oxidative Stress and Cellular Damage

Table 1: Reactive oxygen species (ROS)

Free radicals	Non-radicals	Lipid peroxidation	Secondary products
		products(primary
		products)
Hydroxyl radical:	Hydrogen	Peroxyl	Malondialdehyde
HO·	peroxide:H2O2	radical:ROO·
Superoxide radical:	Singlet oxygen:1O2	Alkoxyl radical:RO·	4-hydroxyalkenals
O.- 2

Free radicals (see Table 1) generated during oxidative stress have many cellular targets. One of the primary targets is cellular lipids (Fig. 3 below)). Lipid peroxidation of polyunsaturated fatty acids (PUFA) results in the formation of peroxyl and alkoxyl radicals (Conklin, 2004). These primary products of lipid  peroxidation, which  are  highly reactive  and relatively short-lived, undergo further reactions to form secondary products of lipid peroxidation that include a variety of aldehydes (Table1) such as malondi- aldehyde, the 4-hydroxyalkenals, and acrolein (Esterbauer et al., 1991). The aldehydes are more stable than the primary products and can diffuse throughout the cell where they damage cellular components and interfere with cellular functions. Because of their electrophilic character, the aldehydes bind to nucleophilic groups of amino acids, such as cysteine, lysine, histidine, serine, and tyrosine, which are critical components of enzyme active sites or are necessary for maintaining the tertiary structure of proteins. The binding of aldehydes

to proteins, which results in enzyme inhibition and alteration of the structure of cellular receptors, may account for the impact of oxidative stress on the cytotoxicity of anti- neoplastic agents (Conklin, 2004).

Fig 3: Production of free radicals via different routes

Source: (Koppeno, 1993)

1.5 Antioxidants

An antioxidant is a molecule that inhibits the oxidation of other molecules (Sies, 1997). Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions produce free radicals and in turn, these radicals start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants have the ability to protect the body from oxidative damage by scavenging the free radicals and inhibiting peroxidation and other radical mediated processes (Ozsoy et al., 2008). They do this by getting oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols (Sies, 1997). Plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, vitamin A, and vitamin E as well as enzymes such as

catalase, superoxide dismutase and various peroxidases.  Antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase etc. protect DNA from oxidative stress. It has been proposed that polymorphisms in these enzymes are associated with DNA damage and subsequently the individual’s risk of cancer susceptibility (Khan et al., 2010).

In recent years, significant attention has been directed towards exploring plant-based natural antioxidants, especially the phenolics and tocopherols (Chaovanalikit and Wrolstad, 2004). Such natural antioxidants are not only reported to have anti-carcinogenic potential that protects the foods from oxidative deterioration but also, these are associated with other health beneficial effects  such as,  lowering the  incidence of aging,  inflammation, cardiovascular diseases and certain cancers (Liu and Yao, 2007). Various antioxidant activity methods have been used to monitor and compare the antioxidant activity of food (Halliwell and Gutterridge, 1999).

Industrially, antioxidants are used as food additives to help guard against food deterioration via oxidation.  Antioxidants  are  frequently  added  to  industrial  products.  A  common  use  is  as stabilizers in fuels and lubricants to prevent oxidation, and in gasolines to prevent the polymerization that leads to the formation of engine-fouling residues (Boozer et al., 1955).

1.6 Classification of Antioxidants

Antioxidant metabolites are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipids (lipophilic) (see Table 2 below). In general, water- soluble antioxidants react with oxidants in the cell cytosol and the blood plasma, while lipid- soluble antioxidants protect cell membranes from lipid peroxidation (Sies, 1997). These compounds may be synthesized in the body or obtained from the diet (Vertuani et al., 2004). The different antioxidants are present at a wide range of concentrations in body fluids and tissues, with some such as glutathione or ubiquinone mostly present within cells, while others such as uric acid are more evenly distributed. Some antioxidants are only found in a few organisms and these compounds can be important in pathogens and can be virulence factors (Miller and Britigan,

1997). The relative importance and interactions between these different antioxidants is a very complex question, with the  various metabolites and  enzyme systems having synergistic and interdependent effects on one another  (Chaudiere and Ferrari-Iliou, 1999). The action of one

antioxidant may therefore depend on the proper function of other members of the antioxidant system (Vertuani et al., 2004). Vertuani et al. (2004) further stated that the amount of protection provided by any one antioxidant will also depend on its concentration, its reactivity towards the particular reactive oxygen species being considered, and the status of the antioxidants with which it interacts. Some compounds contribute to antioxidant defense by chelating transition metals and preventing them from catalyzing the production of free radicals in the cell. Particularly important is the ability to sequester iron, which is the function of iron-binding proteins such as transferrin and ferritin (Imlay, 2003). Selenium and zinc are commonly referred to as “antioxidant nutrients”, but these chemical elements have no antioxidant action themselves and are instead required for the activity of some antioxidant enzymes.

Table 2: Antioxidant metabolites

Antioxidant metabolites	Solubility
Ascorbic acid (vitamin C)	Water
Glutathione	Water
Lipoic acid	Water
Uric acid	Water
Carotenes (vitamin A)	Lipid
α-Tocopherol (vitamin E)	Lipid
Ubiquinol (coenzyme Q)	Lipid

1.6.1 Dietary antioxidants

Ascorbic acid or “vitamin C” is a monosaccharide oxidation-reduction (redox) catalyst found in both animals and plants. As one of the enzymes needed to make ascorbic acid has been lost by mutation during primate evolution, humans must obtain it from the diet; it is therefore a vitamin

(Smirnoff, 2001). As a redox catalyst, vitamin C has antioxidant activity when it reduces, and thereby neutralizes reactive oxygen species such as hydrogen peroxide, however, it will also reduce metal ions that generate free radicals through the Fenton reaction (pro-oxidant activity) (Duarte and Lunec, 2005).

2 Fe3+ + Ascorbate → 2 Fe2+ + Dehydroascorbate

2 Fe2+ + 2 H2O2 → 2 Fe3+ + 2 OH· + 2 OH−

In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the redox enzyme ascorbate  peroxidase,  a  function that  is  particularly  important  in  stress  resistance  in  plants (Shigeoka et al., 2002).

Vitamin E is the collective name for a set of eight related tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties (Herrera and Barbas, 2001). Of these, α- tocopherol has been most studied as it has the highest bioavailability, with the body preferentially absorbing and metabolising this form (Brigelius-Flohé and Traber, 1999). It has been claimed that the α-tocopherol form is the most important lipid-soluble antioxidant, and that it protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction (Herrera and Barbas, 2001). This removes the free radical intermediates and prevents the propagation reaction from continuing. The reaction produces oxidised α-tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol (Wang and Quinn, 1999). This is in line with findings showing that α-tocopherol, but not water-soluble antioxidants, efficiently protects glutathione peroxidase 4 (GPX4)-deficient cells from cell death (Seiler et al., 2008). GPx4 is the only known enzyme that efficiently reduces lipid-hydroperoxides within biological membranes.Beta carotene and other carotenoids (Vitamin A) are also believed to provide antioxidant protection to lipid-rich tissues (Kahkonen et al., 1999). Research suggests that beta carotene may work synergistically with vitamin E (Diplock et al., 1998). A diet that is excessively low in fat may  negatively affect  beta  carotene and  vitamin E  absorption, as  well  as  other  fat-soluble nutrients. 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.

1.6.2 Enzymatic antioxidants

In addition to dietary antioxidants, the body relies on several endogenous defense mechanisms to help protect  against  free  radical-induced cell  damage. The  antioxidant enzymes (glutathione peroxidase, catalase, and superoxide dismutase (SOD)) metabolize oxidative toxic intermediates and require micronutrient cofactors such as selenium, iron, copper, zinc, and manganese for optimum catalytic activity (Okezie, 1996). Intensive agricultural methods have also resulted in significant depletion of these valuable trace minerals in our soils and the foods grown in them (Vlietinck et al., 1995).

Fig 4: Enzymes defense mechanism. Source: (Okezie, 1996)

1.6.2.1 Superoxide dismutase

Superoxide dismutases (SODs) are a class of closely related enzymes that catalyze the breakdown of the superoxide anion into oxygen and hydrogen peroxide (Zelko et al., 2002). SOD enzymes are present in almost all aerobic cells and in extracellular fluids and they contain metal ion cofactors that, depending on the isozyme, can be copper, zinc, manganese or iron (Johnson and Giulivi, 2005). In humans, the copper/zinc SOD is present in the cytosol, while manganese SOD is present in the mitochondrion (Bannister et al., 1987). There also exists a third form of SOD in extracellular fluids, which contains copper and zinc in its active sites (Nozik-Grayck et al., 2005). The mitochondrial isozyme seems to be the most biologically important of these three, since mice lacking this enzyme die soon after birth (Melov et al., 1998). In contrast, the mice lacking copper/zinc SOD (Sod1) are viable but have numerous pathologies and a reduced lifespan, while mice without the extracellular SOD have minimal defects (sensitive to hyperoxia) (Reaume et al.,

1996).

1.6.2.2 Catalase

Catalases are enzymes that catalyse the conversion of hydrogen peroxide to water and oxygen, using either an iron or manganese cofactor and are localized to peroxisomes in most eukaryotic cells (Chelikani et al., 2004). Catalase is an unusual enzyme since (although hydrogen peroxide is its only substrate) it  follows a ping-pong mechanism. Here,  its cofactor is oxidised by one molecule of hydrogen peroxide and then regenerated by transferring the bound oxygen to a second molecule of substrate (Hiner et al., 2002). Despite its apparent importance in hydrogen peroxide removal, humans with genetic deficiency of catalase  (acatalasemia) or mice genetically engineered to lack catalase completely, suffer few ill effects (Ogata, 1991).

1.6.2.3 Glutathione system

The glutathione system includes glutathione (GSH), glutathione reductase (GSR), glutathione peroxidases (GPX) and s-transferase (Meister and Anderson, 1983). This system is found in animals, plants and microorganisms. Glutathione reductase (GR) also known as Glutathione – disulfide reductase (GSR) is an enzyme that catalyzes the reduction of glutathione disulfide (GSSG) to sulfhydryl form glutathione (GSH) which is a critical molecule in resisting oxidative stress and maintaining the reducing environment the cell (Deponte, 2013). GSR functions as

dimeric disulfide oxidoredutase and utilizes FAD prosthetic group and NADPH to reduce one molar equivalent of GSSG to two molar equivalents of GSH. GSH acts as a scavenger for hydroxyl radicals, singlet oxygen and various electrophiles. It reduces oxidized GPX, plays a role in the metabolism and clearance of xenobiotics, acts cofactor to certain detoxifying enzymes, participates in transport, and regenerates antioxidants such as vitamins C and E to their reactive forms. The ratio of GSSG/GSH present in the cell is a key factor in maintaining the oxidative balance of the cell, that is, it is critical that the cell maintains high levels of GSH and low levels of GSSG. This narrow balance is maintained by GSR (Brigelius-Flohé, 1999).

Glutathione peroxidase (GPX) is an enzyme containing four selenium-cofactors that catalyzes the breakdown of hydrogen peroxide and organic hydroperoxides. Therefore, GSR, GPX  and GSH interact to reduce hydrogen peroxide to water, in order to protect the cell from damage. There are at least four different glutathione peroxidase isozymes in animals; Glutathione peroxidase 1 is the most abundant and is a very efficient scavenger of hydrogen peroxide, while glutathione peroxidase 4  is  most  active with  lipid  hydroperoxides (Brigelius-Flohé, 1999). Surprisingly, glutathione peroxidase 1 is dispensable, as mice lacking this enzyme have normal lifespans, but they are hypersensitive to induced oxidative stress (Ho et al., 1997).

Glutathione  S-transferases  (GSTS),  previously  known  as  ligandins,  comprise  a  family  of eukaryotic and prokaryotic phase II metabolic isoenzymes best known for their ability to catalyze the conjugation of GSH to xenobiotic substrates for the purpose of detoxification (Sharma et al.,

2004). In addition, the glutathione S-transferases show high activity with lipid peroxides and these enzymes are at particularly high levels in the liver (Brigelius-Flohé, 1999).

1.7 Review of in vitro antioxidant parameters

It is established that the function of reactive species is highly related to their concentration and the detrimental effects of oxidative stress are linked to the disruption of normal signaling pathways, damage of macromolecules and disruption of homeostasis (Badu et al., 2012). Aristidis et al. (2012) noted that oxygen is not evenly distributed in tissues and cells; therefore, there are sites that are constantly under oxidative stress. Antioxidants present in natural sources help to scavenge free radicals and thus provide health benefits. For these reasons, scientists have embarked on in

vitro studies to establish the antioxidant activity of plant extracts using the parameters discussed below.

1.7.1 The total antioxidant capacity assay

The total antioxidant capacity (TAC) is the term used to describe the ability of antioxidants in different foods to scavenge free radicals in the blood and cells. TAC takes into account the amount of water-based and  fat-based antioxidants present in food. This helps individuals to decide which foods offer the greatest antioxidant benefit. The Total antioxidant capacity by phosphomolybdenum method assay is based on the reduction of Mo (V1) to Mo (V) by the simple analyte and subsequent formation of green phosphate/Mo (V) complex at acidic pH (Raghu et al.,

2011). The phosphomolybdenum method is quantitative since the total antioxidant activity is expressed as the  number of equivalents of ascorbic acid (that  is;  Ascorbic acid equivalents (AAE)) (Prieto et al., 1999).

1.7.2 The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

The model of scavenging the stable DPPH radical is a widely used method to evaluate the free radical scavenging ability of various samples (Lee et al., 2003). Organic free radicals with deep violet color give absorption maxima within 515 and 528nm. Upon receiving proton from any hydrogen donor, mainly from phenolics, it loses its chromophore and becomes yellow (Badu et al., 2012). The phytochemical which might be responsible for the scavenging activity in plant species are the phenolics and flavonoid constituents (Rohman et al., 2010). It is widely accepted that as the concentration of the phenolic compounds increases, DPPH radical scavenging activity and hence antioxidant activity of a plant or a related compound also increases (Sanchez-Moreno et al., 1999). The measurement of the scavenging of DPPH radical allows one to determine exclusively the intrinsic ability of substances to donate hydrogen atom or electrons to this reactive species in a homogenous system. The method is based on the reduction of methanolic-DPPH solution because of the presence of antioxidant substances having hydrogen donating group (RH) such as phenolics and flavonoid compounds due to the formation of a non-radical DPPH-H form (Paixao et al., 2007).

1.7.3 The Reducing Potential Assay (RPA)

The reducing capacity of a compound serves as a significant indicator of its potential antioxidant activity. Extracts of plant source may act as electron donors and they can react with free radicals to convert them into more stable products and terminate radical chain reactions. Also, it has been shown  that  antioxidant  effect  exponentially  increases  as  a  function  of  the  development  of reducing power (Tanaka et al., 1988). The FRAP (Ferric reducing/antioxidant power) assay is

based on the reduction of Fe3+  to Fe2+  by antioxidants in acidic medium (Benzie and Strain,

1996). FRAP is a very simple, rapid and reproducible method which can provide a very useful total antioxidant concentration without measurement and summation of all antioxidants involved.

1.8 Review of hepatic and renal function parameters

1.8.1 The liver

Liver, an important organ actively involved in many metabolic functions, is the frequent target for a  number of toxicants and  hepatic damage  is  associated with distortion of these  metabolic functions (Meyer and Kulkarni et al., 2001). Due to the liver’s ability to the regenerate, even a moderate cell injury is not reflected by measurable change in its metabolic function. However, damage caused by oxidative stress and lipid peroxidation on the membrane of the hepatocytes allows the leakage of some cytoslic enzymes of the liver into the blood stream (Plaa and Hewitt,

1982).

1.8.1.1 Serum enzyme makers of liver disease

When the integrity of the membrane of the hepatocytes is compromised, certain enzymes located in the cytosol are released into the blood. These enzymes are commonly measured clinically as part of a diagnostic evaluation of hepatocellular injury, to determine liver health. Fischbach and Dunning (2004) reported that when used in diagnostics, it is usually measured in international units/litre (iu/L). Liver enzymes include Alanine aminotransferase (ALT), Aspartate aminotransferase (AST)  and  Alkaline  phosphatase (ALP).  ALT,  also  called  serum glutamic pyruvate transaminase (SGPT) is found in serum and  in various bodily tissues, but  is most commonly associated with the liver (Pagana and Pagana, 2010). While sources vary on specific normal range values, Kaplan (2002) reports 5-60 iu/L as the normal range for SGPT. Significantly

elevated levels of ALT (SGPT) often suggest the existence of medical problems such as liver damage, heart failure, viral hepatitis, bile duct problems, infectious mononycleosis or myopathy (Gelfand and Steinberg, 1977).

AST,  also  called  serum glutamic oxaloacetate transferase (SGOT) is  a  pyridoxal phosphate (PLP)-dependent transaminase enzyme that catalyses the reversible transfer of α- amino group between  aspartate  and  glutamate,  and  such  as  such,  an  important  enzyme  in  amino  acid metabolism (Kirsch et al., 1984). AST is similar to alanine aminotransferase (ALT) in that both cells are associated with liver paranchymal cells (Hayashi et al., 1990). The difference is that ALT  is  found  predominantly in  the  liver,  with  clinically  negligible quantities  found  in  the kidneys, heart and skeletal muscle, while  AST is found in the liver, heart, skeletal muscle, kidneys and brain and red blood cells (Gelfand and Steinberg, 1977). As a result, ALT is more specific indicator of liver inflammation than AST, as AST may be elevated also in diseases affecting other organs (Mcphalen et al., 1992). Gaze (2007) reported that the reference range of SGOT is 8-40 iu/L and 6-14 iu/L for male and female respectively. ALP is present in all tissues throughout the entire human body, but is particularly concentrated in the liver, intestine, bile ducts, bone and  the placenta (Kim and  Wyckoff, 1991). The  normal range is  44-147 iu/L, although high levels of ALP are seen in children undergoing growth spurts and in pregnant women (Martin, 2011). Significantly elevated levels show liver damage and may also signify that the bile ducts are blocked.

1.8.2   Serum urea

Urea is a by-product from metabolism of proteins in the liver, regulated by N-acetylglutamate, dissolved in the blood (in reference range of 2.5 and 6.7 mmol/litre) and is excreted by the kidney as a component of urine (Clarkson et al., 2008; Deepak et al., 2007). In addition, a small amount of urea  is excreted in sweat. Urea serves an important role  in the  metabolism of nitrogen- containing compounds and is synthesized in the body of many organisms as part of the urea cycle, either from oxidation of amino acids or from ammonia (Deepak et al., 2007) The blood urea test is a measure of the amount of nitrogen in blood in the form of urea and is a measurement of renal function (Clarkson et al., 2008). Deepak et al. (2007) reported that normal adult human blood should contain between 7-21mg of urea nitrogen per 100ml (7-21mg/dl) of blood.

1.8.3 Serum creatinine

Creatinine is the breakdown product of creatine phosphate in the muscle and is usually produced at a fairly constant rate by the body (Israni and Kasiske, 2011). It is a chemical made by the body and is used to supply energy mainly to muscles. Creatinine blood test is done to ascertain how well the kidneys work (the test is an indicator of renal function). Creatinine is removed from the body by the kidneys and if the kidneys are malfunctioning, creatinine levels increases in the blood (because less creatinine is released through the urine). Israni and Kasiske (2011) reported that creatinine level also varies according to an individual’s size and muscle mass. The normal range for men is 0.7-1.3 mg/dl and 0.6 – 1.1 mg/dl for women (Israni and Kasiske, 2011).

1.9 Haematological studies

Haematology is a  medical specialty that  deals  with blood, blood-forming organs and  blood diseases (Emejulu, 2013). It includes the study of aetiology (causes), diagnosis, treatment, prognosis  (prediction  of  possible  outcome)  and  prevention  of  blood  diseases.  Blood  is  a specialized body fluid. It is composed of pale yellow fluid called plasma in which are suspended red cells (erythrocytes), white cells (leukocytes) and platelets (thrombocytes). Plasma forms about

55% of blood volume and contains water (95%) and many solutes, including proteins, mineral ions,  organic  molecules,  hormones,  enzymes,  products  of  digestion  and  waste  products  of excretion (Emejulu, 2013). Most times, haematological studies are carried out to investigate anaemia and monitor a person’s response to treatment, investigate infections and pyrexia (fever) of  unknown  origin  (PUO),  investigate  haemoglobinopathies (genetic  defects  that  results  in abnormal structure of one of the globin chains of haemoglobin molecule) which are clinically important   (Emejulu,   2013).   According   to   Emejulu   (2013),   there   are   about   5-6   litres (approximately 10 pints) of blood in the circulatory system of an adult (7-8% of a person’s body weight), and about 300 ml of blood in the system of a newborn infant.

1.9.1 Red blood cells (Erythrocytes)

RBCs are the most common type of blood cell and are the principal means of delivering oxygen from  the  lungs  or  gills  to  body tissues  through the  blood.  Erythrocytes consist  mainly  of haemoglobin, a complex molecule containing heme groups whose iron atoms temporarily link to oxygen  molecules.  Anaemia  which  is  characterized  by an  abnormally  low  concentration of

haemoglobin  is  one  of the  most  common blood diseases (Cohen,  1982).  One  of the  major symptoms of anaemia is fatigue due to the failure of RBC to transport enough oxygen to the body tissues (Synder and Brandon, 1999). When insufficient iron is available to the bone marrow, it slows down its production of haemoglobin and RBCs (iron-deficient anaemia). Adult humans

have roughly 2 – 3 x 1013 RBCs at any given time (Snyder and Brandon, 1999).

1.9.2 White blood cells (Leucocytes)

WBCs or leucocytes make up about one percent (1%) of blood in a healthy adult and they play a role in the body’s immune system primary defence mechanism against invading bacteria, viruses, fungi and parasites (Dacie and Lewis, 1991). White blood cell count is the estimation of the total number  of  WBCs  per  liter   in  blood.  High  WBC  counts  come  with  acute  infections, inflammations, trauma and cancer such as leukemia while low blood cell counts may be caused by problems with their production or with autoimmune disease, where the body fights its own cells mistakenly or after viral infections (Shah and Altindag, 2004). There are about five differential WBC namely; Lymphocytes, Neutrophil, Eosinophil, Monocytes and Basophils. The normal range for the WBC count (leukocyte count) varies between laboratories but is usually between 4,300 and 10,800 cells per cubic millimeter of blood and can be expressed as 11× 109/ L(Dacie and Lewis, 1991).

1.9.3 Haemoglobin Haemoglobin (Hb) is the iron-containing oxygen transport metaloprotein in the red blood cells of vertebrates (Hardison, 1996). In mammals, the proteins make up about 97% of the dry content of red cells and about 35% of the total content (including water). Haemoglobin transports oxygen from the lungs or gills to the rest of the body, such as to the muscles, where it releases the oxygen for cell use. It also has variety of other roles of gas transport and effect-modulation which vary from species to  species and are quite diverse in some vertebrates (Eshaghian et  al., 2006). Decrease  in  haemoglobin  with  or  without  an absolute decrease of red  blood cell,  leads  to symptoms of Anaemia which have different causes. Haemolysis (accelerated breakdown of red blood cells), associated with jaundice is caused by increased blood concentration of bilirubin (a haemoglobin  metabolite)  and  the  circulating  bilirubin can  cause  renal  failure.  Haemoglobin concentration measurement is one of the most common blood tests performed, usually as part of a complete blood count. For  example,  it  is  usually tested before blood donation.  Results are reported in g/L, g/dl, or mol/l. 1g/l equals about 0.6206 mmol/l and normal values are: 12.1 to 15.1 g/dl for women, 13.5 to 16.5 g/dl for men, 11 to 16 g/dl for children and 11 to 12 g/dl for pregnant women (Eshaghian et al., 2006).

1.9.4 Packed cell volume (PCV)

PCV (also referred to as haematocrit or erythrocyte volume fraction) is the volume percentage of packed red blood cells found in 100ml of blood and it is normally 45% and 40% for adult males and females respectively (Purves et al., 2004). PCV can be used as a screening tool for anaemia and can also indicate the degree of fluid loss during dehydration. Decrease in PCV is mostly due to  acute/chronic blood  loss and  immune  mediated diseases and  it  usually  indicates  internal hemorrhage before any other symptoms become apparent (Karlow et al., 1969). PCV is increased usually during abnormal polycythemia (increase in RBCs) and dehydration (Gustric and Pearson, 1982).

1.9  Review on Histopathology

Histopathology (from the Greek histos (tissue) and pathos (suffering)) refers to the microscopic examination of tissues in order to study the manifestation of disease (Kniep et al., 2015) Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. This is the most important tool of the anatomical pathologist in routine clinical diagnosis of cancer and other diseases. Histopathological examination of tissues starts with surgery, biopsy or autopsy (Kniep et al., 2015). The tissue is removed and then placed in a fixative which stabilizes the tissue to prevent decay. The most common fixative is formalin (10% formaldehyde in water). The histological slides are examined under a microscope by a pathologist, a medically qualified specialist. The diagnosis is formulated as a pathology report describing the histological findings and the opinion of the pathologist.

1.11 Aim of the study

The aim of this research was to investigate the antioxidant effect of the flavonoid-rich fraction of the methanol extracts of Jatropha tanjorensis leaves on some in vitro antioxidant models and CCl4-induced hepatotoxicity in rats, so as to verify its claims.

1.12 Specific objectives of the study

  Quanlitative and quantitative phytochemical screening of the methanol extract of Jatropha tanjorensis leaves.

  Extraction /  solvent-solvent  partitioning  of the  pulverized plant  leaves  to  obtain  the flavonoid-rich fraction of the plant leaves.

  Determination of total flavonoid concentration (TFC) of the ethylacetate-soluble fraction.

  Determination of total antioxidant capacity (TAC) of the flavonoid-rich fraction of the plant leaves.

  Assessment of Ferric reducing antioxidant power (FRAP) of the flavonoid-rich fraction of the plant leaves.

  Assessment of radical scavenging activity of the flavonoid-rich fraction of the plant leaves

(DPPH radical scavenging activity).

  Toxicity study (LD50) of the flavonoid-rich fraction of Jatropha tanjorensis leaves.

  Determination  of  effect  of  the  flavonoid-rich  fraction  of  the  plant  leaves  on  some enzymatic antioxidants (SOD, Catalase and Glutathione peroxidase).

  Determinations of effect of the flavonoid-rich fraction of the plant leaves on liver marker enzymes and on kidney function parameters (urea and creatinine).

  Evaluation  of  effect  of  the  flavonoid-rich  fraction  of  the  plant  leaves  on  some haematological parameters.

  Effect of the flavonoid-rich fraction of the plant leaves on the histology of the Liver of rat models.

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