TOXIC PROFILE OF THE METHANOL LEAF EXTRACT OF SENNA SIAMEA AND ITS ANTIOXIDANT PRPOPERTIES

ABSTRACT

Medicinal plants have been assumed to be good in detoxifications because they contain bioactive compounds capable of doing this. However, some plants are very toxic to both humans and animals with the potential of damaging certain organs in the body. Many plant- derived  substances,  collectively termed  “phytochemicals,”  are  becoming  increasingly known for their antioxidant activity. This study is aimed at investigating the toxic and as well as the antioxidant profile of methanol extract of Senna siamea leaves, extensively used in Nigeria folk medicine to manage various ailments. The potent metabolites were quantified using standard methods and the antioxidant scavenging potentials of the extract on DPPH and superoxide radicals were also determined. Both acute and sub-acute toxicity profile were evaluated by determining the LD50, liver and kidney function tests, which was then correlated and confirmed with histopathologic techniques of hepatocytes and kidney cells.  Furthermore,  haematological  parameters  of the test  rats  were  determined.  The qualitative phytochemical screening of the leaves of S. siamea revealed the presence of proteins, carbohydrates, tannins, alkaloids, steroids, glycosides, flavonoids, reducing sugars, terpenoids and quinones in the leaves. The result of these tests also indicates that tannin, carbohydrates, reducing sugar, terpenoids, alkaloids, total phenols, glycosides and flavonoids were contained in various amounts when quantified. The antioxidant vitamin contents of the extract was also quantified and this showed that vitamin E was significantly higher (p< 0.05), (181.70±2.47 mg/100g) when compared to vitamin A (40.35±13.60 mg/100g) and vitamin C (4.11±0.06 mg/100g). The extract scavenged DPPH and superoxide radicals in concentration dependent manner. The EC50 of DPPH., superoxide radical were 12.59±0.00 and 39.38±0.01 respectively. Acute toxic test shows no death was observed in any group of the mice used, indicating that the extract could be tolerated by the mice at 5000mg/kg bw, the highest concentration used. The liver marker enzymes AST and ALP showed significantly increased (p<0.05) in their serum activity while ALP activity showed no significant changes (p>0.05) when compared to the control. Furthermore, bilirubin concentration were significantly increased (p<0.05) during the 3rd and 4th week of administration of extract to the 4th group. The kidney function test showed a significant increase (p<0.05) in creatinine concentration was observed in the first and second week. A significant increase (p<0.05) in serum urea concentration was observed in the 1st  week of group 4. The result of the blood electrolytes showed  no significant difference (p>0.05) in sodium ion but significant changes (p<0.05) were observed in the levels of Potassium and chloride ion. The hematological parameters (White Blood Cell, Hemoglobin and packed cell volume) did not produce any significant changes (p>0.05). Significant reduction (p< 0.05) was observed in catalase activities of the 2nd and 3rd weeks of extract administration. These results obtained indicate that the extract could be toxic to hepatocytes and kidney cells at higher concentration.

CHAPTER ONE

INTRODUCTION

Medicinal plants are believed to be important sources of new chemical substances with potential

therapeutic effects. Their activities depend on its phytochemical contents (Meyer et al., 1997). Presently, it is estimated that about 80% of the world’s population are dependent on medicinal plants or plant-derived products for their health needs (Shri, 2003). The use of medicinal plant is presently on the increase due to its availability, affordability, accessibility, and promising efficacy comparable to the often high cost and adverse effects associated with standard synthetic drug agents (Kwada and Tella, 2009). One therefore expect bioactive compounds obtained from such plants to have low animal and human toxicity. However, people are often unaware of important similarities and differences between medicinal herbs and approved medications (Williamson et al.,

1996). Some mistakenly think of herbs as natural alternatives to chemicals, failing to recognize that herbs are composed of bioactive chemicals, some of which may be toxic (Fabricant and Farnsworth, 2001). Medicinal plants have been documented to have advantage in toxicity considerations based on their long term use (Zhu et al., 2002).Some plants are very toxic to both humans and animals with the potential of damaging certain organs in the body. (Edoga et al.,

2005).  This calls for caution in the use of medicinal plants. Toxicity studies on medicinal plants or extracts usually determine the level of safety particularly during the development of drugs (Jaijoy et al., 2010).The use of traditional and herbal medicine is widely practiced in Nigeria and Senna siamea is one of such plants used in Nigerian folklore medicine. It is hence necessary to evaluate the safety of the use of S. siamea in wistar rats.

1.1       Senna siamea

1.1.1    Taxonomy and Nomenclature

Domain:         Eukaryota Kingdom:     Plantae Phylum:        Spermatophyta Subphylum: Angiospermae Class:            Dicotyledonae Order:           Fabales

Family:        Fabaceae Subfamily:   Caesalpinioideae Genus:         Senna

Species:       Senna siamea

1.1.2    Botanical description, Ecology and cultivation

Senna siamea also called Cassia siamea is an angiosperm native of Southeast Asia and widely distributed in Africa, Latin America and in Oceania (Sosef et al., 1998). It is a medium-size, evergreen tree growing up to 18 m tall with a straight trunk of up to 30 cm in diameter. This plant is a shrub which has a medium-size, 10-12m tall, occasionally reaching 20 m (Jensen, 1995). The bole is short; crown dense and rounded at first, later becoming irregular and spreading. The young bark is grey and smooth, and later with longitudinal fissures. The leaves are alternate, 15-30 cm long, compound, with 6-14 leaflets each ending in a tiny bristle and the flowers are bright yellow, in large, up to 60 cm long, upright, with pyramid-shaped panicles (Sosef et al., 1998). The root system consists of a few thick roots, growing to considerable depth, and a dense mat of rootlets in the top 10-20 cm of soil, which may reach a distance of 7 m from the stem in 1 year and eventually a distance up to 15 m (Sahni, 1981). The fruits are flat with indehiscent pod, 5-30cm long, and constricted between the seeds. There are about 20 seeds per pod. S. siamea starts flowering and fruiting at the age of 2-3 years. Once established, it flowers precociously and abundantly throughout the year.  The seeds are bean-shaped, greenish-brown, and 8-15 mm long (Gutteridge,

1997). Ecologically, S. siamea grows in a range of climatic conditions but is particularly suited to lowland tropics with a monsoon climate. It grows only when its roots have access to ground water, and the maximum length during dry period usually do not exceed 48 months (Sahni, 1981).

1.1.3 Uses of Senna siamea

1.1.3.1 Therapeutic Uses

The leaves, stems, roots, flowers and seeds of S. siamea regardless the subspecies, have been used for the treatment of several ailments including mostly malaria, a tropical endemic disease with high mortality (Shivjeet et al., 2013). According to the ethnic differences in populations from localities, the plant is used alone or in combination with other plants or natural substances for the preparation especially in decoction (Ejobi et al., 2007; Maurya and Dongarwar, 2012). The leaves are the most used part of the plant especially by African and Asian population in preparation of the herbal remedies. In Nigeria, the dried leaves are mixed and boiled with lemon (Cymbopogon citratus), pawpaw (Carica papaya), and lime leaves (Citrus lemonum). The “tea” of the mixture is drunk against malaria (Ogunkunle and Ladejobi, 2006). Other authors reported that S. siamea leaves decoction is drunk against constipation and hypertension and are inhaled in toothache

(Otimenyi et al., 2010). Furthermore, Aliyu (2006), revealed that S. siamea is ethno-medicinally used as laxative, blood cleaning agent, cure for digestive system and urogenital disorders (Smith,

2009). According to Otimenyin et al., 2010, a traditional claim have cited Senna siamea to be used for the treatment of typhoid fever, jaundice, abdominal pain and menstrual pain. In addition, it is claimed to be used for reducing sugar level in the blood (Ngamrojanavanich et al., 2006). Senna siamea has been shown to be effective in managing constipation associated with a number of causes including surgery, childbirth and the use of narcotic pain relievers (Mohammed et al., 2012). According to scientific support with regard to therapeutic efficacy, commercially available tablets of S. siamea leaf powder have been very popular in Thailand for producing natural sleep. However, use for this purpose is now discouraged due to hepatotoxicity (Padumanonda et al., 2007). The flowers and young fruits are regularly consumed as vegetable and for treating curries. It provides laxative and sleeping-aid effect (Kiepe, 1995).

1.1.4.2 Economic and Industrial Uses

The wood is dense and excellent for fuel. The foliage is used for green manure and fodder for cattle, sheep and goats but is toxic for pigs and poultry. The species is also used for erosion control, land reclamation (including abandoned mine areas), shade, shelter and ornamentals (Maurya and Dongarwar, 2012). In Nigeria, it has been used as a planted fallow crop and to reclaim abandoned tin-mined areas (Ekeleme et al., 2005).

1.2       Toxicity: An Overview

Toxicity is the degree to which a substance (a toxin or poison) can harm humans or animals (Fauci and Anthony, 2008). According to Cutler, 2010, the toxicity of a substance depends on the following: form and innate chemical activity, dosage, especially dose-time relationship, exposure route, species, age, sex, ability to be absorbed, metabolism, distribution within the body, excretion and presence of other chemicals. Toxic effects are generally categorized according to the site of the toxic effect (Larrey, 1997). In some cases, the effect may occur at only one site and this site is referred to as the specific target organ. In other cases, toxic effects may occur at multiple sites and this is referred to as systemic toxicity. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a

cell (cytotoxicity) or an organ (organotoxicity), such as the liver (hepatotoxicity). Excess of any compound will be harmful to life and considered under toxicity studies.

1.2.1    Liver: Morphology, Functions and Toxicity

The liver is the first major organ to be exposed to ingested chemicals due to its portal blood supply (Naruse et al., 2007). Although chemicals are delivered to the liver to be metabolized and excreted, this can frequently lead to activation of liver injury (Larrey, 1997). Functions of the liver include biotransformation of xenobiotics, endogenous compounds, including hormones carbohydrate metabolism and storage synthesis of blood proteins (albumin, lipoproteins) urea formation, fat metabolism and bile formation (Willett et al., 2004). It is also involved in the synthesis of products like glucose derived from glycogenesis, plasma proteins, clotting factors and urea that are released into the bloodstream (Navarro, 2006). It regulates blood levels of amino acids (Lindor et al., 2009). It is also involved in the production of a substance called bile that is excreted to the intestinal tract (Larrey, 1997). Hepatotoxicity refers to liver dysfunction or liver damage that is associated with an overload of drugs or xenobiotics (Kedderis, 1996). Hepatotoxicants are exogenous compounds of clinical relevance and may include overdoses of certain medicinal drugs, industrial chemicals, and natural chemicals like herbal remedies and dietary supplements (Willett et al., 2004). Hepatotoxicity related symptoms may include a jaundice, severe abdominal pain, nausea or vomiting, weakness, severe fatigue, continuous bleeding, skin rashes, generalized itching, swelling of the feet and/or legs, abnormal and rapid weight gain in a short period of time, dark urine and light colored stool (Singh et al., 2011). The liver enzyme tests, formerly called liver function tests (LFTs), are a group of blood tests that detect inflammation and damage to the liver (Teschke,

2009). They can also check how well the liver is working. When liver cells are damaged or destroyed, the enzymes in the cells leak out into the blood, where they can be measured by blood tests (Singh et al., 2011). Enzymes used in checking for liver damage includes ALT, AST and alkaline phosphatase (Amacher, 2002). Study of the liver has been and continues to be important in understanding fundamental molecular basis of toxicity as well as in assessment of risks to humans (Saukkonen et al., 2006). The cause of the hepatotoxicity of S. siamea capsules is still unexplained. In 2001, Chivapat and his colleagues studied the subchronic effect of barakol (a major constituent of S. siamea leaves) in rats and blood biochemistry parameters were investigated. The results revealed that S. siamea capsules caused degeneration and necrosis of hepatocytes in Wistar rats and the severity of the lesion was dose dependent.

1.2.1.1 Alanine Aminotransferases

The standard clinical biomarker of hepatotoxicity, alanine aminotransferase (ALT) activity is the most frequently relied biomarker of hepatotoxicity (Ruhl and Everhart, 2012). It is a liver enzyme that plays an important role in amino acid metabolism and gluconeogenesis. It catalyzes the reductive transfer of an amino group from alanine to α-ketoglutarate to yield glutamate and pyruvate (Amacher, 2002). Elevated level of this enzyme is released during liver damage (Ruhl and Everhart, 2012). The estimation of this enzyme is a more specific test for detecting liver abnormalities since it is primarily found in the liver (Amacher, 2002). However, lower enzymatic activities are also found in skeletal muscles and heart tissue. This enzyme detects hepatocellular necrosis (Ruhl and Everhart, 2012).

1.2.1.2 Aspartate Aminotransferase

Aspartate aminotransferases (AST) catalyzes the reductive transfer of an amino group from aspartate to α-ketoglutarate to yield oxaloacetate and glutamate (Ozer et al., 2008). Besides liver, it is also found in other organs like heart, muscle, brain and kidney. Injury to any of these tissues can cause an elevated blood level (Nathwani et al., 2005). It also helps in detecting hepatocellular necrosis but is considered a less specific biomarker enzyme for hepatocellular injury as it can also signify abnormalities in heart, muscle, brain or kidney (Ozer et al., 2008; Dufour et al., 2000). The ratio of serum AST to ALT can be used to differentiate liver damage from other organ damage (Nathwani et al., 2005).

1.2.1.3 Alkaline Phosphatase

Alkaline phosphatase (ALP) is a hydrolase enzyme that is eliminated in the bile and it hydrolyzes monophosphates at an alkaline pH (Dufour et al., 2000). It is particularly present in the cells which line the biliary ducts of the liver and may be elevated if bile excretion is inhibited by liver damage (Wright and Vandenberg, 2007). It is also found in other organs including bone, placenta, kidney and intestine (Ramaiah, 2007). Hepatotoxicity leads to elevation of the normal values due to the body’s inability to excrete it through bile due to the obstruction of the biliary tract, which may occur within the liver, the ducts leading from the liver to the gallbladder, or the duct leading from the gallbladder through the pancreas that empty into the duodenum (Ozer et al., 2008).

1.2.1.4 Total Bilirubin

Serum bilirubin levels are also used to check liver damage (Singh et al., 2011). Bilirubin is an endogenous anion derived from the regular degradation of haemoglobin from the red blood cells and excreted from the liver in the bile (Geuken et al., 2004). It is measured as total bilirubin and direct bilirubin. Total bilirubin is a measurement of all the bilirubin in the blood while direct bilirubin is a measurement of a water-soluble conjugated form of bilirubin made in the liver (Dufour et al., 2000). Indirect bilirubin is calculated by the difference of the total and direct bilirubin and is a measure of unconjugated fraction of bilirubin (Ozer et al., 2008). When the liver cells are damaged, they may not be able to excrete bilirubin in the normal way, causing a buildup of bilirubin in the blood and extracellular (outside the cells) fluid (Saukkonen et al., 2006). Increased levels of bilirubin may result due to decreased hepatic clearance which may lead to jaundice and other hepatotoxicity symptoms (Thapa and Walia, 2007).

1.2.2    Kidney: Morphology, Functions and Toxicity

The kidneys serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and regulation of blood pressure (via maintaining salt and water balance) (Bauer et al., 1982). Nephrotoxicity, also referred to as renal toxicity, is the development of functional or structural kidney damage after exposure to one or more of a wide variety of drugs, other treatments or exogenous toxins, and may lead to a variety of functional consequences and structural lesions (Thomas, 2005). Otimenyin and his colleagues, 2010, that worked on the pharmacological basis of the root of S.siamea observed that the treated groups showed no effect in renal failure as there was no significant increase in urea and creatinine when the extract was administered to the wistar rats. When kidney damage occurs, the kidney will be unable to rid the body of excess urine and wastes (Sangu et al., 2011). The blood electrolytes (such as potassium and magnesium) become elevated as well. Elevated levels of BUN and/or creatinine may be due to a temporary condition such as dehydration or a sign of renal failure (Garabed et al., 1997).

1.2.2.1 Creatinine

Creatinine is a metabolite of phosphocreatine (pcreatine), a molecule used as a store for high- energy phosphate that can be utilized by tissues for the production of ATP (Gault, 1976). Creatine either comes from the diet or synthesized from the amino acids arginine, glycine, and methionine (Wyss and Kaddurah-Daouk, 2000). Creatine and p-creatine are converted non-enzymatically to the metabolite creatinine, which diffuses into the blood and is excreted by the kidneys (Wallimann, et al., 1992). Approximately 2% of the body’s creatine is converted to creatinine every day and it is transported through the bloodstream to the kidneys (Gault, 1976). The kidneys filter out most of the creatinine and dispose of it in the urine. Creatinine has been found to be a fairly reliable indicator of kidney function and elevated creatinine level signifies impaired kidney function or kidney disease. Additionally altered creatinine levels may be associated with other conditions that result in decreased renal blood flow such as diabetes and cardiovascular disease (Ferreira et al.,

2005). As the kidneys become impaired for any reason, the creatinine level in the blood will rise due to poor clearance of creatinine by the kidneys (Wallimann, et al., 1992).

1.2.2.2 Blood Urea Nitrogen (BUN)

Blood urea nitrogen (BUN) level is another indicator of kidney function (William et al., 1995). In the kidneys, the contribution of urea to medullary hypertonicity is an important determinant of the ability of the kidneys to generate concentrated urine (Levey et al., 1999). Urea is also a metabolic by-product which can build up if normal kidney function is impaired. Serum blood urea nitrogen (BUN) levels, however, may provide supplemental information in regard to renal function as renal proximal tubule cells may increase BUN reabsorption in the setting of increased neuro-hormonal activation (Shlipak et al., 2002). The BUN-to-creatinine ratio generally provides more precise information about kidney function and its possible underlying cause compared with creatinine level alone (Levey et al., 1999).

1.2.2.3 Blood Electrolytes

Electrolytes, such as sodium, potassium, magnesium and chloride are substances (macro minerals) that become ions in solution and acquire the capacity to conduct electricity (Clayman, 1989). Electrolytes are present in the human body, and the balance of the electrolytes in the body is essential for normal function of the cells and the organs. A depletion of electrolytes in the body can cause fatigue, cramping, and risk of dehydration. Replacing electrolytes is essential to keep the body hydrated, to enhance performance, restore balance, and help prevent muscle cramps (Aukland and Nicolaysen, 1981). In addition, electrolytes play an important part of the body processes that are essential for living, such as muscle function, heart function, and nerve reaction (Horne and Swearingen, 1993). Electrolytes regulate the acidity (pH) in the blood. It is important to ensure electrolytes are in balance to provide restorative health to the body and create a sense of balance and wellness (Clayman, 1989).

1.2.2.3.1          Chloride Ion

Chloride is the major anion (negatively charged ion) found in the fluid outside of cells and in the blood (Aukland and Nicolaysen, 1981). Chloride ion also plays a role in helping the body maintain a normal balance of fluids (Allison, 2004) and the balance of chloride ion (Cl-) is closely regulated by the body (Awad et al., 2008). Significant increases or decreases in chloride can have deleterious or even fatal consequences (Edelman and Leibman, 1959). Elevations in chloride ion may be seen in diarrhea and certain kidney diseases (Handy and Soni, 2008). Chloride is normally lost in the urine, sweat, and stomach secretions (Horne and Swearingen, 1993). Excessive loss can occur from heavy sweating, vomiting, adrenal gland and kidney disease (Edelman and Leibman, 1959).

1.2.2.3.2          Sodium Ion

Sodium is the major positive ion (cation) in fluid outside of cells (Awad et al., 2008). Excess sodium (such as that obtained from dietary sources) is excreted in the urine (Trinder, 1951). Sodium regulates the total amount of water in the body and the transmission of sodium into and out of individual cells also plays a role in critical body functions (Allison, 2004). Many processes in the body, especially in the brain, nervous system, and muscles, require electrical signals for communication (Flear and Singh, 1982). The movement of sodium is critical in generation of these electrical signals (Trinder, 1951). Therefore, too much or too little sodium can cause cells to malfunction, and extremes in the blood sodium levels (too much or too little) can be fatal (Aukland

and Nicolaysen, 1981). Increased sodium (hypernatremia) in the blood occurs whenever there is excess sodium in relation to water (Allison, 2004). There are numerous causes of hypernatremia; these may include kidney disease, too little water intake, and loss of water due to diarrhea and/or vomiting (Awad  et  al.,  2008).  A decreased  concentration  of sodium  (hyponatremia) occurs whenever there is a relative increase in the amount of body water relative to sodium (Flear and Singh, 1982). This happens with some diseases of the liver and kidney, in patients with congestive heart failure, in burn victims, and in other numerous conditions (Trinder, 1951).

1.2.2.3.3          Potassium Ion

Potassium ion is the major positive ion (cation) found inside of cells. The proper level of potassium is essential for normal cell function (Terri and Sesin, 1958).   Among the many functions of potassium in the body are regulation of the heartbeat and the function of the muscles (Aukland and Nicolaysen, 1981). A seriously abnormal increase in potassium (hyperkalemia) or decrease in potassium (hypokalemia) can profoundly affect the nervous system and increases the chance of irregular heartbeats (arrhythmias), which, when extreme, can be fatal (Terri and Sesin, 1958). Potassium is normally excreted by the kidneys, so disorders that decrease the function of the kidneys can result in hyperkalemia (Rose and Post 2001). Hypokalemia, or decreased potassium, can arise due to kidney diseases; excessive loss due to heavy sweating, vomiting, or diarrhea, eating disorders, certain medications, or other causes (Allison, 2004) .

1.3       Antioxidants and Free Radicals

1.3.1    Antioxidant

Antioxidant is a substance that has the ability to delay or prevent the oxidation of a substrate by inhibiting the initiation or propagation of oxidizing chain  reactions caused by free radicals (Bahorun et al., 2006). Oxidation reactions can produce free radicals, which start chain reactions that damage cells (Halliwell, 1997). The antioxidant process can function in one of two ways: chain-breaking or prevention. For the chain-breaking, when a radical releases or steals an electron, a second radical is formed. The last one exerts the same action on another molecule and continues until either the free radical formed is stabilized by a chain-breaking antioxidant (vitamin C, E, carotenoids and so on), or it simply disintegrates into an inoffensive product (Valko et al., 2007). The classic example of such a chain reaction is lipid peroxidation. For the preventive way, an

antioxidant enzyme like superoxide dismutase, catalase and glutathione peroxidase can prevent oxidation by reducing the rate of chain initiation, example, either by scavenging initiating free radicals or by stabilizing transition metal radicals such as copper and iron (Halliwell, 2007). Antioxidants terminate these chain reactions by removing radical intermediates and inhibiting other oxidation reactions by being oxidized themselves (Lunec and Duarte, 2005).  Low levels of antioxidants or inhibition of the antioxidant enzymes causes oxidative stress and may damage or kill cells (Valko et al., 2007). The antioxidant defense systems function through blocking the initial production of free radicals, scavenging the oxidants, converting the oxidants to less toxic compounds, blocking the secondary production of toxic metabolites or inflammatory mediators, blocking the chain propagation of the secondary oxidants, repairing the molecular injury induced by free radicals or enhancing the endogenous antioxidant defense system of the target (Sati et al.,

2010). These defense mechanisms act cooperatively to protect the body from oxidative stress. The antioxidant defense system consists of powerful enzymatic and non-enzymatic antioxidants (Halliwell, 2007).

1.3.1.1 Enzymatic Antioxidants

All cells in the body contain powerful antioxidant enzymes. The three major classes of antioxidant enzymes are the superoxide dismutases, catalases and glutathione peroxidases. In addition, there are numerous specialized antioxidant enzymes reacting with and detoxifying oxidants (Valko et al., 2007).

1.3.1.1.1          Superoxide Dismutases (SODs)

They are a class of closely related enzymes that catalyse the breakdown of the superoxide anion into oxygen and hydrogen peroxide (Zelko et al., 2002). They are present in almost all aerobic cells and in the extracellular fluids. They contain metal ions that can be copper, zinc, manganese or iron. In humans, the copper/zinc superoxide dismutase is present in the cytosol, while manganese superoxide dismutase is present in the mitochondria. There also exists a third form of superoxide dismutase in extracellular fluids, which contains copper and zinc in its active sites (Johnson and Giulivi, 2005). Superoxide dismutase removes O2–  by catalyzing a dismutation

reaction. In the absence of superoxide dismutase, this reaction occurs non-enzymatically but at a very slow rate (Nozik-Grayck et al., 2005).

1.3.1.1.2          Catalase

Catalase (H2O2 oxidoreductase) is a tetramer of four polypeptide chains, each over 500 amino acids long, contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide (Ho et al., 2004). It can decompose hydrogen peroxide (H2O2) in reactions catalyzed by two different modes of enzymatic activity: the catalytic mode of activity (2H2O2→O2 + 2H2O) and the peroxidatic mode of activity (H2O2 + AH2→A + 2H2O) (Parejo et al., 2002). Catalase is an unusual enzyme since, although hydrogen peroxide is its only substrate, it follows a ping-pong mechanism (Mayo et al., 2003). Here, its cofactor is oxidized by one molecule of hydrogen peroxide and then regenerated by transferring the bound oxygen to a second molecule of substrate (Kabel et al., 2013). Catalase is present in all prokaryotes and eukaryotes (Yang et al., 2003). Since H2O2 serves as a substrate for certain reaction that generate the highly reactive hydroxyl radical, catalase is believed to play a role in cellular antioxidant defense mechanisms by limiting the accumulation of H2O2 (Mayo et al., 2003). Overexpression of catalase renders cells more resistant to toxicity of H2O2  and oxidant-mediated injury (Kabel et al., 2013). Decreases in the levels of catalase signifies increased oxidative stress and reduced antioxidant activities in the system which reduces the capability of the body to get rid of free radicals (Parejo et al., 2002).

1.3.1.2 Antioxidant Vitamins

Antioxidants from diet play important role in helping endogenous antioxidants for the neutralization of oxidative stress (Halliwell, 1997). The nutrient antioxidant deficiency is one of the causes of numerous chronic and degenerative pathologies. Each nutrient is unique in terms of its structure and antioxidant function (Donaldson, 2004).

1.3.1.2.1          Vitamin E

Vitamin E is a fat-soluble vitamin with high antioxidant potency. Vitamin E is a chiral compound

with eight stereoisomers: α, β, γ, δ tocopherol and α, β, γ, δ tocotrienol. Only αtocopherol is the

most bioactive form in humans (Miller et al., 2005). Studies in both animals and humans indicate that natural D-α-tocopherol is nearly twice as effective as synthetic racemic DL-α-tocopherol (Nguyen et al., 2006). Because it is fat-soluble, αtocopherol safeguards cell membranes from damage by free radicals. Its antioxidant function mainly resides in the protection against lipid peroxidation (Halliwell, 1997). Vitamin E has been proposed for the prevention against colon, prostate and breast cancers, some cardiovascular diseases, ischemia, cataract, arthritis and certain neurological disorders (Sen et al., 2006; Miller et al., 2005).

1.3.1.2.2          Vitamin C

Vitamin C also known as ascorbic acid, is a water-soluble vitamin. It is essential for collagen, carnitine and neurotransmitters biosynthesis (Li and Schellhorn, 2007). Health benefits of vitamin C include antioxidant, anti-atherogenic, anti-carcinogenic and immunomodulatory functions (Naidu, 2003). The positive effect of vitamin C resides in reducing the incidence of stomach cancer, and in preventing lung and colorectal cancer (Halliwell, 1997). Vitamin C works synergistically with vitamin E to quench free radicals and also regenerates the reduced form of vitamin E (Li and Schellhorn, 2007). Natural sources of vitamin C are acid fruits, green vegetables and tomatoes. Ascorbic acid is a labile molecule, therefore it may be lost from diet during cooking (Naidu, 2003).

1.3.1.2.3          Beta-carotene

Beta-carotene is a fat soluble member of the carotenoids which are considered pro-vitamins because they can be converted to active vitamin A. Beta-carotene is converted to retinol, which is essential for vision (Halliwell, 1997). It is a strong antioxidant and is the best quencher of singlet oxygen. Beta-carotene is present in many fruits, grains, oil and vegetables (carrots, green plants, squash, spinach) (Willcox et al., 2004).

1.3.2    Free Radicals

Free radicals are continuously produced by the body’s normal use of oxygen (Tiwari, 2004). When cells use oxygen to generate energy, free radicals are produced by the mitochondria. These by- products are generally reactive oxygen species (ROS) as well as reactive nitrogen species (RNS)

that result from the cellular redox process. Free radicals have a special affinity for lipids, proteins, carbohydrates and nucleic acids (Velavan, 2011). A free radical is any chemical species (capable of independent existence) possessing one or more unpaired electrons; an unpaired electron being one that is alone in an orbital. The simplest radical is the hydrogen atom. Electrons are more stable when paired together in orbital: the two electrons in a pair have different directions of spin. Hence, radicals are generally less stable than non-radicals, although their reactivity varies. Free radicals are capable of reacting indiscriminately with any molecule with which they come in contact. Once radicals are formed, they can either react with another radical or with another non-radical molecule by various interactions (Tiwari, 2004). If two radicals collide they can combine their unpaired electrons, thus forming a covalent bond. However, most molecules found in vivo are non-radicals. In this case a radical might donate its unpaired electron to the other molecule, or might take one electron from it, thus transforming its radical character. At the same time a new radical is formed (Halliwell, 1997).

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