ABSTRACT
Stephania dinklagei is used extensively in South East Nigeria for the traditional treatment of malaria and other associated ailments in form of decoction, in which unspecified quantities are usually consumed without due regards to toxicologic and other adverse effects. In this study, the phytochemicals were assessed as well as the effects of the antioxidant and toxicologic properties of methanol leaf extract of stephania dinklagei in Wistar albino rat. The rats were administered with graded doses of the extract twice daily for three weeks and the control administered with distilled water. Four rats each from the control and test groups were sacrificed every seven days and blood samples collected for analysis. The percentage yield of stephania dinklagei methanol leaf extract was 5.5%. Preliminary phytochemical screening showed that the methanol leaf extract contained alkaloids, flavonoids, tannins, steroids, terpenoids, carotenoids, glycosides, anthocyanins and saponins. Anthraquinone was not detected. The quantitative phytochemical analysis showed that the extract contains alkaloids (29.70 + 0.15mg/g), flavonoids (25.30 + 0.10mg/g), steroids (69.70 + 0.10mg/g), saponins (13.57 + 0.21mg/g), tannins (64.21+ 0.21mg/g) cardiac glycosides (1.45 +
0.09mg/g), terpenoids (44.30 + 0.26mg/g), carotenoids (5.88 + 0.52mg/g) and anthocyanin
(15.40 + 0.26mg/g). The vitamin content of the leaf extract was found to be vitamin A (44.8
+ 0.42mg/100g), vitamin C (27.85 + 0.07mg/100g) and vitamin E (12.7 + 0.28mg/100g). The acute toxicity test of the leaf extract showed no toxicity up to 5000mg/kg body weight as observed over a period of 48 hrs for signs of acute toxicity. The extract was found to moderately scavenge the DPPH and superoxide anion radical in a dose dependent manner compared with their respective standards. The extract however, highly scavenged the hydroxyl radical when compared with the standard, α-tocopherol. There were no significant differences (p >0.05) in serum MDA level in all the groups in week I but significantly increased (p<0.05) in group 4 (week 2) when compared with that of their control. The serum SOD activity showed a significant decrease (p<0.05) in all the groups of 1st, 2nd and 3rd weeks
of the experiment when compared with that of their respective controls. Serum CAT also decreased significantly (p<0.05) in group 3 and 4 in week 3 compared with the control but no significant difference (p<0.05) was observed in all the groups in week 1 and 2. Serum ALP activity increased significantly (p<0.05) throughout the duration of the experiment when compared with that of their controls. Serum ALT level increased significantly (p<0.05) only
in group 4 in the 1st, 2nd and 3rd weeks of the experiment. The same trend was observed with
the AST level when compared with those of their controls. Creatinine showed a non- significant increase (p>0.05) in groups 2 and 3 but significantly decreased (p<0.05) in group
4 (week 1). There were also non-significant difference (p>0.05) in all the groups in week 2 when compared with that of their control but in week 3, there was non-significant increase (p>0.05) in groups 2 and 3 and a non-significant decrease in group 4. Urea level significantly increased (p<0.05) in all the groups throughout the duration of the experiment. Serum Na+ increased significantly (p<0.05) in week 1, 2 and 3 compared with those of their respective controls. Serum Cl- level showed non-significant difference (p>0.05) in week 1 and 2 but however, increased significantly (p<0.05) in week 3 compared with the control. Histological examination of the liver cells of the treated rats revealed widespread hepatocellular vacuolar degeneration with hypertrophy of kupffer cells in the periportal areas and moderate infiltration of mononuclear leucocytes into the periportal area as against that of their control. The histopathology result corroborates the results of the serum biochemical parameters. The kidney showed no significant changes in the treated groups compared with that of their
control. These results suggest that Stephania dinklagei leaf extract had a significant in vitro antioxidant activity. However, long term consumption of the extract at the doses studied could be hepatotoxic but not nephrotoxic.
CHAPTER ONE
INTRODUCTION
The Southern inhabitants of Nigeria are known for their high consumption of vegetables. Some of these vegetables form part of foods consumed on special conditions, including ill health and times of convalescence. This stresses the role of plants in the life of man (Nwangwu et al., 2009). The use of plant parts in traditional medical practice has a long drawn history and remains the mainstay of primary health care in most of third world countries (Prescott-Allen, 1982). Medicinal plants are believed to be an important source of some secondary metabolites with potential therapeutic benefits (Farnsworth, 1989). In treatment of diseases, the use of herbs has gained grounds world wide, making traditional medicine an inevitable global discuss. This practice calls for research into pharmacological activities of plants secondary metabolites and has improved modern pharmaco therapeutics around the world (Nwaogu et al., 2007). Though, some medicinal plants serve as food, they contain secondary metabolites that influence biological processes and reverse disease states (Ugochukwu and Badaby, 2002).
In normal or pathological cell metabolism, free radicals which have one or more unpaired electrons are produced. Reactive Oxygen Species (ROS) react easily with free radicals such as superoxide anion radical (O2-) and hydroxyl anion (OH-) as well as non-free radical species
(H2O2) and the singlet oxygen (1O2) (Yildrim et al., 2002). Also excessive generation of ROS
induced by various stimuli and which exceed the antioxidant capacity of the organism leads to a variety of pathophysiological processes such as inflammation, diabetes, genotoxicity and cancer (Kourounakis et al., 1999). A great number of medicinal plants contain chemical compounds that exhibit antioxidant properties (Gulcin et al., 2002). Sources of natural antioxidants are primarily plant phenolics that may occur in all parts of plant such as fruits, vegetables, nuts, seeds, leaves and barks (Pratt and Hudson, 1990).
1.1 Stephania dinklagei
Figure 1: Stephania dinklagei leaves
A large number of alkaloids have been isolated from Stephania dinklagei, it contains corydine (sedative drug agent) and Stephanini (analgesic drug agent) (Goren et al., 2003). Its constituent Liriodenine exhibits antiprotozoal and cytotoxic activities against the protozoa Leishmania donovania and Plasmodium falciparum (Camacho et al., 2000). An infusion made from its young leaves is immediately given to children before it thickens to relieve them from stomach aches (Goren et al., 2003). Its leaves are taken to treat impotency in men and also act as an aphrodisiac (Burkill, 1997). Given its uses in the traditional setting and the emerging reports on its pharmacological actions, it is therefore necessary to evaluate its antioxidant properties, toxicity and its effect on liver and kidney marker enzymes in Wistar albino rats.
1.2 Phytochemicals
Phytochemicals are chemical compounds formed during the plants normal metabolic processes. These chemicals are often referred to as secondary metabolites which include alkaloids, flavonoids, coumarins, glycosides, gums, polysaccharides, phenols, tannins, terpenes and terpenoids (Harborne, 1973; Okwu, 2004). These can act as agents to prevent undesirable side effects of the main active substances or to assist in the assimilation of main substances (Anonymous, 2007). Phytochemicals are present in a variety of plants utilized as important components of both human and animal diets. These include fruits, seeds, herbs and vegetables (Okeke and Elekwe, 2003). Most of these phytochemical constituents are potent bioactive compounds found in medicinal plant parts which are precursors for the synthesis of useful drugs (Sofowora, 1993).
1.2.1 Alkaloid
Alkaloids are group of naturally occurring low molecular weight nitrogenous chemical compound that contain mostly basic nitrogen atoms (Manske, 1965). They are found primarily in plants and are especially common in certain families of flowering plants (Herbert, 1999). Large variety of organisms produce alkaloids, these include bacteria, fungi, plants and animals and are part of the group of natural products called secondary metabolites (Baldwin and Ohnmeiss, 1993). Most alkaloids contain oxygen in their molecular structure, those compounds are usually colourless crystals at ambient conditions (Lewis and Elvin- Lewis, 1977). Oxygen-free alkaloids, such as nicotine or coniine are typically volatile, colourless, oily liquids (Abuo-Donia et al., 1992) some alkaloids are coloured, like berberine (yellow) and sanguinarine (orange) (Akhtar et al., 2003). Most alkaloids are weak bases, but some, such as theobromine and theophylline are amphoteric (Ali and Khan, 2008). Many alkaloids dissolve poorly in water but readily dissolve in organic solvents such as diethyl ether, chloroform or 1, 2-dichloroethane. Caffeine, cocaine, codeine and nicotine are water soluble (Ashihara et al., 2008). Biological precursors of most alkaloids are amino acids such as ornithine, lysine, phenylalanine, tyrosine, tryptophan, histidine, aspartic acid and anthranilic acid (Berkov et al., 2007). Alkaloid biosynthesis are too numerous and cannot be easily classified (Blankenship et al., 2005).
Most of the known functions of alkaloids are related to protection. For example, aporphine alkaloid – liriodenine produced by the tulip tree protects it from parasitic mushrooms. Many alkaloids are used in medicine: Atropine, Codeine, Nicotine and Quinine reserpine are used as anticholinergic, stimulant; antipyretics and antihypertensives respectively (Ashihara et al.,
2008).
1.2.2 Saponin
Saponins are amphipathic glycosides grouped, in terms of phenomenology, by the soap-like foaming they produce when shaken in aqueous solutions (Francis et al., 2002). Saponins consist of a polycyclic aglycones attached to one or more sugar side chains. The aglycone part, which is also called sapogenin is either steroid (C27) or a triterpene (C30) (Skene,
2006). The foaming ability of saponins is caused by the combination of a hydrophobic (fat- soluble) sapogenin and a hydrophilic (water soluble) sugar part. Saponins have a bitter taste. Some saponins are toxic and are known as sapotoxin (XU et al., 1996). They are found in most plants, vegetables, beans and herbs (Francis et al., 2002). Studies have illustrated the beneficial effects on blood cholesterol levels, cancer, bone health and stimulation of the immune system (XU et al., 1996). It has also shown that saponins have anti tumor and anti- mutagenic activities and can lower the risk of human cancers by preventing cancer cells from
growing. It was found that saponins may help to prevent colon cancer and as shown in their article “Saponins as anti-carcinogens” published in the Journal of Nutrition (1995).
1.2.3 Steroid
Steroids are organic compounds that contain a characteristic arrangement of four cycloalkane rings that are joined to each other (Kuzuyama and Seto, 2003). Examples of steroids include the dietary fat cholesterol, the sex hormones estradiol and testosterone and anti-inflammatory drug dexamethasone (Rosier, 2006). Steroids are found in plants, animals and fungi. All steroids are made in cells either from the sterols lanosterol (animals and fungi) or from cycloartenol (plants). Both lanosterol and cycloartenol are derived from the cyclization of the triterpene squalene (Kuzuyama and Seto, 2003). Steroids have a chemical structure that contains the core of gonane or a skeleton derived there from. Usually, methyl groups are present at the carbons C-10 and C–13 – an alkyl side-chain at carbon C–17 may also be
present (Dubey, et al., 2003).
H R
H H
H H
Fig. 2: The basic skeleton of a sterHoid, with standard stereo orientation
The three cyclohexane rings form the skeleton of phenanthrene, the last ring of the gonane has a cyclopentane structure. Hence, together they are called cyclopentaphenanthrene (Hanukoglu, 1992). Steroid biosynthesis is an anabolic metabolic pathway that produces steroids from simple precursors. A unique biosynthetic pathway is followed in animals compared to many other organisms, making the pathway a common target for antibiotics and anti-infective drugs (Hanukoglu, 1992). In addition, steroid metabolism in humans is the target of cholesterol lowering drugs such as statins. In humans and other animals, the biosynthesis of steroids follows the mevalonate pathway that uses acetyl-CoA as building blocks to form dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) (Dubey et al, 2003). In subsequent stages, DMAPP and IPP are joined to form geranyl
pyrophosphate (GPP), which in turn is used to synthesize the steroid lanosterol, lanosterol can then be converted into other steroids such as cholesterol and ergosterol (Rosier, 2006).
1.2.4 Flavonoid
Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms. Flavonoids consist of 6 major subgroups: Chalcone, flavone, flavonol, flavanone, anthocyanins and isoflavonoids. Together with carotenes, flavonoids are also responsible for the colouring of fruits, vegetables and herbs (Galeotti et al., 2008).
Flavonoids are synthesized by the phenyl-propanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA (Cazarolli et al., 2008; Friedman,
2007). This can be combined with malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones, which contain two phenyl rings. Conjugate ring- closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone (Friedman, 2007). The metabolic pathway continues through a series of enzymatic modifications to yield flavanones →diydroflavonols→anthocyanins. Along this pathway, many products can be formed, including the flavonols, flavan-3-ols, proanthocyanidins (tannins) and a host of other various polyphenolics (Verueridis et al., 2007). In vitro studies show that flavanoids also have anti-allergic, anti-inflammatory, anti-microbial, anti-cancer and anti-diarrheal activities (Cushnie and Lamb, 2011). Research has shown that flavonoids are poorly absorbed in the human body (less than 5%), with what is absorbed being quickly metabolized and excreted (Williams et al., 2004).
1.2.5 Tannins
Tannin is an astringent, bitter plant polyphenolic compound that binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids (Muller- Harvey and McAllan, 1992). Tannin compounds are found mainly in plants where they play a role in protection from predation, and perhaps also as pesticides (Giner-Chavez, 1996). Tannins play an important role in the ripening of fruit and the aging of wine. There are three large classes of secondary metabolites in plants: Nitrogen containing compounds, terpenoids and phenolics (Mole, 1993). Tannins belong to the phenolics class. All phenolic compound (primary and secondary) are, in one way or another formed via the shikimic acid pathway, also known as the phenylpropanoid pathway (Reed, 1995). The same pathway leads to the formation of other phenolics such as isoflavones, coumarins, lignins and aromatic amino acids (tryptophan, phenylalnie and tyrosine). Typically, tannin molecules require at least twelve hydroxyl groups and at least five phenyl groups to function as protein binders.
Tannins are important ingredient in the process of tannin of leather. Tannins produce different atoms with ferric chloride according to the type of tannin (Souza et al., 2006).
1.2.6 Anthocyanin
Anthocyanins are water-soluble vacuolar pigments that belong to a parent class of molecules called flavonoids synthesized via the phenylpropanoid pathway, they are odourless and nearly flavourless (Stafford, 1994). Anthocyanins occur in all tissues of higher plants including leaves, stems, roots, flowers and fruits (Wada and Ou, 2002). Anthocyanins have been shown to act as a “sunscreen”, protecting cells from high-light damage by absorbing blue-green and ultraviolet light, thereby protecting the tissues from photo inhibition, or high-light stress (Lieberman, 2007). Anthocyanins can be used as pH indicators because their colour changes with pH; they are pink in acidic solutions, purple in neutral solutions, greenish-yellow in alkaline solutions and colourless in very alkaline solutions where the pigment is completely reduced (WU et al., 2004). They are found in cell vacuole. The anthocyanins are subdivided into the sugar-free anthocyanidin aglycones and the anthocyanin glycosides. As of 2003, more than 400 anthocyanins had been reported (Lieberman, 2007). While more recent literature (early 2006) puts number at more than 550 different anthocyanins. The difference in chemical structure that occurs in response to changes in pH is the reason why anthocyanins are often used as pH indicators, as they change from red in acids to blue in bases (De-Rosso et al., 2008). In anthocyanin biosynthetic pathway, L-phenylalanine is converted to naringenin by phenylalanine ammonialyase (PAL), Cinnamate-4-hydroxylase (C4H), 4- Coumarate CoA Ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI). And then, the next pathway is catalysed by the formation of complex aglycone and anthocyanin
composition by flavone 3 – hydroxylase (F3H), flavonoid 31 – hydroxylase (F31H),
dihydroflavonol 4 – reductase (DFR). Anthocyanidin synthase (ANS). UDP – glucoside: flavonoid glucosyltransferase (UFGT) and methyl transferase (MT). Among these, UFGT is divided into UF3GT and UF5GT, which are responsible for the glucosylation of anthocyanin to produce stable molecules (WU et al., 2004). Although anthocyanins are powerful antioxidants in vitro, this antioxidant property is unlikely to be conserved after the plant is consumed (Stafford, 1994).
1.2.7 Cardiac Glycoside
Cardiac glycosides are glycosides of mostly C23 – steroidal compounds. They have a characteristic 5 – or 6 – membered lactone ring (Wang et al., 2008). They are called cardiac glycosides because they modify heart action (Brower et al., 1972). Cardenolides inhibit the
Na+ – K + – ATPase pump in mammals. This group of compounds is found in a large number
of families many of which are unrelated. A number of toads and frogs make cardiac active compounds that are steroidal but not glycosidic in nature (Wang et al., 2008). Cardenolides are derived from steroidal precursors, probably cholesterol via the intermediacy of pregnenolone or progesterone intermediates (Jungreis et al., 1997). Most members of the family Asclepiadaceae contain cardiac glycosides (Dussourd, 1986). Cardiac glycosides are drugs used in the treatment of congestive heart failure and cardiac arrhythmia (Dussourd,
1986). Drugs such as Ouabain and digoxin are cardiac glycosides. Digoxin from the foxglove plant is used clinically, whereas Ouabain is used only experimentally due to its extremely high potency (Dussourd, 1986). Normally, sodium-potassium pumps in the membrane of cells (in this case, cardiac myocytes) pump potassium ions in and sodium ions out. Cardiac glycosides inhibit this pump by stabilizing it in the E2 – P transition state, so that sodium cannot be extruded: intracellular sodium concentration increases. A second membrane ions exchanger, NCX, is responsible for pumping calcium ions out of the cell and sodium ion in (3Na/Ca): raised intracellular sodium levels inhibit this pump, so calcium ions are not extruded and will also begin to build up inside the cell (Jungreis et al., 1997).
1.2.8 Terpenoid
Terpenoid are large and diverse class of naturally occurring organic chemicals similar to terpenes derived from five-carbon isoprene units assembled and modified in thousands of ways (Yousefbeyk et al., 2014). Most are multicyclic structures that differ from one another not only in functional groups but also in their basic carbon skeletons (Wolinsky, 1973). These lipids can be found in all classes of living things and are the largest group of natural products (Gimelli, 2001). The steroids and sterols in animals are biologically produced from terpenoid precursors. Sometimes terpenoids are added to proteins, e.g. to enhance their attachment to the cell membrane, this is known as isoprenylation (Maarse, 1991). Terpenoids can be thought of as modified terpenes, wherein methyl groups have been moved or removed or oxygen atoms added (Swan, 1967). Just like terpenes, the terpenoids can be classified according to the number of isoprene units used. There are two metabolic pathways of creating terpenoids: Mevalonic acid pathway and MEP/DOXP pathway (Wolinsky, 1973).
1.2.9 Carotenoid
Carotenoids are organic pigments that are found in the chloroplasts of plants and some other photosynthetic organisms like algae, some bacteria and some fungi (Armstrong and Hearst,
1996). Carotenoids can be produced from fats and other basic organic metabolic building blocks by all these organisms (Brian, 1991).
Carotenoids are split into two classes, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons and contain no oxygen) (Unlu et al., 2005; Brian, 1991). The major role of carotenoid in plants and algae is that they absorb light energy for use in photosynthesis and they protect chlorophyll from photo damage (Kidd, 2011). Carotenoids belong to the category of tetraterpenoids, structurally, carotenoids take the form of a polyene hydrocarbon chain which is sometimes terminated by rings and may or may not have additional oxygen atoms attached (Unlu, et al., 2005). The most common carotenoids include lycopene and the vitamin A precursor, B-carotene. In plants, the xanthophylls lutein is the most abundant carotenoid and its role in preventing age-related eye disease is currently under investigation (Alija, et al., 2004).
1.3 Antioxidants and free radicals
Antioxidants are molecules which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged (Vertuani et al., 2004). It is therefore an oxidation reaction (Davies, 1995). Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions (Stohs and Bagchi, 1995). Antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols (Valko et al., 2007). Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules (Knight, 1998; Stohs and Bagchi,
1995). Once formed, these highly reactive radicals can start a chain reaction. Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane (Valko et al., 2004). Cells may function poorly or die if this occurs. To prevent free radicals damage, the body has a defense system of antioxidants (Benzie, 2003; Davies, 1995). A paradox in metabolism is that, while the vast majority of complex life on earth requires oxygen for its existence, oxygen is highly reactive molecule that damages living organisms by producing reactive oxygen species (Valko et al.,
2007). Consequently, organisms contain a complex network of antioxidants metabolism and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids (Jha et al., 1995). In general, antioxidant systems either prevent these reactive species from being formed or remove them before they can damage vital components of the cell (Sies, 1997; Davies, 1995). 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. 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
(O2-) (Hirst et al., 2008). The hydroxyl radical is particularly unstable and will react rapidly and non-specifically with most biological molecules (Jha et al., 1995). This species is produced from hydrogen peroxide in metal-catalyzed redox reactions such as the Fenton reaction (Sies, 1997). These oxidants can damage cells by starting chemical chain reactions
such as lipid peroxidation, or by oxidizing DNA or proteins (Vertuani et al., 2004). Damage to DNA can cause mutations and possibly cancer, if not reversed by DNA repair mechanisms (Knight, 1998) while damage to protein causes enzyme inhibition, denaturation and protein
degradation (Davies, 1995).
Ischemic injury Nephrotoxic injury Phagocyte activation HOCl Phagocyte activation
NADPH oxidize Xanthine oxidase Mitochondria
Cl MPO
Fe2+ Fe3+
•O2+e- →O2•- H2O2 OH• X
Lipid peroxidation
L-arg.→ NO+.
INOS
GSSG GSH
Figure 3: Production and elimination oHf OR+OOS (Hirst et al., 2008)
Ishemic
ONOO- 2 2
1.3i.n1juryHydroxyl radical
The hydroxyl radical, .HO, is the neutral form of the hydroxide ion (HO-). Hydroxyl radicals are highly reactive and consequently short-lived; however, they form an important part of radical chemistry (Sies, 1993). Hydroxyl radicals are produced from the decomposition of hydroperoxides (ROHO) or atmospheric chemistry by the reaction of excited atomic oxygen with water (Reiter and Caneiro, 1997), Hydroxyl radicals are also produced during UV-light dissociation of H2O2 and likely in Fenton chemistry, where trace amounts of reduced transition metals catalyze peroxide – mediated oxidations of organic compounds (Sunil et al.,
2013; Reiter and Caneiro, 1997). The hydroxyl radical is often referred to as the “detergent” of the troposphere because it reacts with many pollutants often acting as the first step to their removal (Sies, 1993). It has an important role in eliminating some greenhouse gases like methane and ozone (Storey et al., 1981). The first reaction with many volatile organic
compounds (VOCs) is the removal of an hydrogen atom, forming water and an alkyl radical
(R•) (Sies, 1993) •HO + RH → H2O + R•
The alkyl radical will typically react rapidly with oxygen forming a peroxy radical R• + O2
→ RO2 (Sunil et al., 2013). Hydroxyl radicals can occasionally be produced as a byproduct of immune action. Macrophages and microglia most frequently generate this compound when exposed to very specific pathogens, such as certain bacteria (Sies, 1993; Sunil et al., 2003). The destructive action of hydroxyl radicals has been implicated in several neurological autoimmune diseases such has HAND when immune cells become over-activated and toxic to neighbouring healthy cells (Sies, 1993). The hydroxyl radical can damage virtually all types of macromolecules (Sies, 1993; Storey et al., 1981). Unlike superoxide, which can be detoxified by superoxide dismutase, the hydroxyl radical cannot be eliminated by an enzymatic reaction (Storey et al, 1981). Mechanisms for scavenging peroxyl radicals for the protection of cellular structures includes endogenous antioxidants such as melatonin and glutathione and dietary antioxidants such as mannitol and vitamin E. (Reiter and Carneiro, 1997).
1.3.2 Superoxide anion radical
It has the chemical formula O2• -. It is the product of one-electron reduction of molecular (O2), which occurs widely in nature (Holleman and Wibers, 2001). Superoxide is biologically quite toxic and is deployed by the immune system to kill invading microorganisms (Muller et al.,
2007). In phagocytes, superoxide is produced in large quantities by the enzyme NADPH oxidase for use in oxygen-dependant killing mechanisms of invading pathogens (Miller and Fridovich, 1986). Mutations in the gene coding for the NADPH oxidase cause an immune deficiency syndrome called chronic granulomatous disease (Muller et al., 2007; Rapoport et al., 1994) characterized by extreme susceptibility to infection, especially catalase positive organisms (Rapoport et al., 1994). Because superoxide is toxic, nearly all organisms living in the presence of oxygen contain isoforms of the superoxide scavenging enzyme, superoxide dismutase (SOD) (Holleman and Wibers, 2011). SOD is an extremely efficient enzyme that catalyzes the neutralization of superoxide nearly as fast as the two can diffuse together spontaneously in solution (Rapoport et al., 1994).
1.4 Dietary antioxidants
Dietary antioxidants vitamins C, E and beta carotene are among the most widely studied vitamins and are group of organic substance present in minute amounts in foods stuffs that are essentially for normal metabolism (Bender, 2003; Kutsky, 1973; Halliwel 1991). Vitamins also directly scavenge ROS and upregulate the activities of antioxidant enzymes
(Topinka et al., 1989). 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, 1997). Vitamin E is one of the most important antioxidants; it inhibits ROS – induced generation of lipid peroxyl radicals thereby protecting cells from peroxidation of PUFA in membrane phospho-lipids from oxidative damage of plasma very low density lipoprotein, cellular proteins, DNA and from membrane degeneration (Aruoma,
1998). Consequently, a dietary deficiency of vitamin E reduces the activities of hepatic catalase, GSH peroxidases and glutathione reductase (Fischer – Nielson et al., 1992). Vitamin C has been cited as being capable of regenerating vitamin E (Sies, 1997). Beta carotene and other carotenoids are believed to provide antioxidant protection to lipid – rich tissues. Research suggests beta carotene may work synergistically with vitamin E (Sies, 1997). 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 (Sies, 1997).
1.5.0 Oxidative stress
As remarkable as our antioxidant defense system is, it may not always be adequate. Oxidative stress reflects an imbalance between the systematic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage (Sies, 1997; Finkel and Holbrook, 2000). Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA (Bjelakovic et al.,
2007; Benzie, 2003). Oxidative stress can cause disruptions in normal mechanisms of cellular
signaling. Reactive oxygen species can be beneficial as they are used by the immune system as a way to attack and kill pathogens (Stohs and Bagchi, 1995). Short-term oxidative stress may also be important in prevention of aging by induction of a process named mitohormesis (Bjelakovic et al., 2007). Oxidative stress is associated with increased production of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses, such as glutathione (Vertuani et al., 2004). The effects of oxidative stress depend upon the size of these changes, with a cell being able to overcome small perturbations and regain its original state (Valko et al., 2004). Oxidative damage in DNA can cause cancer. Several antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxide, 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 (Valko et al., 2004; Sies, 1997). The inflammatory response that occurs after strenuous exercise is also associated with oxidative stress (Knight,
1998). During this process, free radicals are produced by neutrophils to remove damaged tissue. As a result, excessive antioxidant levels may inhibit recovery and adaptation mechanisms (Davies, 1995). Antioxidants supplements may also prevent any of the health gains that normally come from exercise, such as increased insulin sensitivity (Knight, 1998;
Benzie, 2003).
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