EFFECT OF ETHANOL EXTRACT OF DENNETTIATRIPETALA ON LIVER AND KIDNEY ANTIOXIDANT ENZYME ACTIVITY AND MALONDIALDEHYDE CONCENTRATION OF ALBINO WISTAR RATS EXPOSED TO CCL4.


EFFECT OF ETHANOL EXTRACT OF DENNETTIATRIPETALA ON LIVER AND KIDNEY ANTIOXIDANT ENZYME ACTIVITY AND MALONDIALDEHYDE CONCENTRATION OF ALBINO WISTAR RATS EXPOSED TO CCL4.  

ABSTRACT

The effect of ethanol extract of Dennettiatripetala on rats exposed to carbon tetrachloride was investigated.  Ethanol extract of the plant was prepared using standard procedure. Sets of 30 female wistar albino rats were divided into 6 groups containing five animals each and were treated orally with increasing doses of ethanol extract of Dennettiatripetala for two weeks. CCl4 was diluted with olive oil in a 1:1 ratio and administered once by oral route at the end of the extract administration. Results from the study showed non- significant decreases in the levels of catalase and SOD activities (P>0.05) in the CCl4 group compared to the control. The extract treatment however produced a higher activity for the antioxidant enzymes compared to the CCl4treated groups. The results also showed increased levels of MDA concentration (P<0.05) in the CCl4 group whereas extract treated rats showed lower concentrations of MDA. The overall results suggests that the ethanolic extract of Dennettiatripetala may have moderate hepatoprotective effect in the CCl4 induced rats.

TABLE OF CONTENT

Title page ……………………………………………….……………………i

Certification………………………………………………………………….ii

Dedication……………………………………………………………………iii

Acknowledgement……………………………………………………………iv

Table of contents………………………………………..……………………v

Abstract……………………………………………………………………….vi

CHAPTER ONE

1.0 INTRODUCTION……………………………………………………… 1

1.1LITERATURE REVIEW……………………………..…………………..3

1.1.0 THE LIVER……………………………………………………………..3

1.1.0.1 FUNCTIONS OF THE LIVER …………………..………………….4

1.1.0.2 BIOTRANSFORMATION OF HEPATOTOXICANTS…………….5

1.2 KIDNEY…………………………………………………………………..7

1.2.1 FUNCTIONS OF THE KIDNEY………………………………………7

1.2.1.0 EXCRETION OF WASTES…………………………………………..8

1.2.1.1 REABSORPTION OF VITAL NUTRIENTS……..………………….8

1.2.1.2 ACID-BASE HOMEOSTASIS……………………………………….9

1.2.1.3 OSMOLALITY REGULATION………………………………….….9

1.2.1.4 BLOOD PRESSURE REGULATION…………………….……….10

1.2.1.5 HORMONE SECRETION…………………………………………..11

1.3 HEPATOTOXICITY………………………………………..…………..12

1.3.1 SYMPTOMS OF HEPATOTOXICITY…………………..…………..13

1.3.1.0 CLINICAL MANIFESTATION…………………………………….13

1.3.1.1 PATHOLOGICAL MANIFESTATION…………………….………14

1.4 CARBONTETRACHLORIDE………………………………………….14

1.5 OXIDATIVE STRESS…………………………………………………..18

1.5.1 EFFECT OF OXIDATIVE STRESS ON DNA……………………….18

1.5.2 EFFECT OF OXIDATIVE STRESS ON LIPIDS…………………….20

1.5.3 EFFECT OF OXIDATIVE STRESS ON PROTEINS………..……….20

1.5.4 LIPID PEROXIDATION……………………………………..………..21

1.6 ANTIOXIDANTS………………………………………………………..25

1.6.0 ENZYMATIC ANTIOXIDANTS……………………………………...25

1.6.0.1 SUPEROXIDE DISMUTASE………………………………………..25

1.6.0.2 CATALASE………………………………………………………….26

1.6.0.3 GLUTATHIONE PEROXIDASE…………………………………...27

1.6.1 NON ENZYMATIC ANTIOXIDANTS……………………………….29

1.6.1.1 VITAMIN C…………………………………………………………..29

1.6.1.2 VITAMIN E………………………………………………………….29

1.6.1.3 GLUTATHIONE…………………………………………………….30

1.7 LIVER FUNCTIONTESTS………………………………………………30

1.8 LIPID PROFILE…………………………………………………………33

1.9 DENNETTIA TRIPETALA……………………………………………............34

JUSTIFICATION…………………………………………………………….44

AIM AND OBJECTIVE……………………………………………………..44

CHAPTER TWO……………………………………………………………..45

2.0 MATERIALS AND METHODS…………………………………………45

2.1 MATERIALS…………………………………………………………..…45

2.2 APPARATUS……………………………………………………………46

2.3 METHODS ………………………………………………………………47

2.3.1 EXPERIMENTAL ANIMALS………………………………………....47

2.3.2 COLLECTION AND EXTRACTION OF PLANTMATERIALS…….47

2.4 ANIMAL FEEDING EXPERIMENT……………………………………48

2.5 CATALASE……………………………………………………………...49

2.6 SUPEROXIDE DISMUTASE……………………………………………50

2.7 MALONDIALDEHYDE………………………………………………….51

CHAPTER THREE

3.0 RESULTS……………………………………………………….53

CHAPTER FOUR

DISCUSSION……………………………………………………….58

CONCLUSION………………………………………………………66

REFERENCES………………………………………………………..67

APPENDIX I……………………………………………………….. 75

APPENDIX II………………………………………………………...76

APPENDIX III………………………………………………………77

APPENDIX IV………………………………………………………79

APPENDIX V……………………………………………………..…81

APPENDIX VI………………………………………………………84

APPENDIX VII………………………………………………………89

CHAPTER ONE

1.0  INTRODUCTION

            Natural plant products and their derivatives represent more than 50% of all the drugs in clinical use in the world (Ben-Eric, 2002). Dennettiatripetalaalso known as pepper fruit tree is a well-known Nigerian spicy medicinal plant.  It is found in the tropical rainforest region of Nigeria and sometimes in Savanna areas (Okwu et al., 2005).  It is locally called “Nkarika” by the Efiks of Calabar.  The young leaves and fruits have distinctive spicy taste.  The mature fruits constitute the main edible portions.  Some communities in parts of Southern Nigeria also utilize the leaves and roots, in addition to the fruits for medicinal purpose. Dennettiatripetalahas been found tocontain lots of minerals, vitamins, alkaloids and trace elements which are of medicinal importance. It was also indicated that the rich presence of essential oil (oleoresins) determines the aromatic flavoring, coloring and pungent properties of pepper fruits. (Nwaoguet al., 2007) investigated phytochemical content of  Dennettiatripetala and detected the presence of saponins, flavonoids, tannins and cyanogenic  glycosides. The intake of flavonoids in any fruit and vegetable tends to decrease cancer risk (Neuhouser, 2004; Grafet al., 2005). Flavonoid contributes to the color of plants, their fruits and flowers. The use of medicinal plants in traditional medicine is not intended in any way to replace modern medical science but rather an aid in conventional therapy (Ben-Eric, 2002).

Carbon tetrachloride (CCl4) is an industrial chemical that does not occur naturally. Most of the carbon tetrachloride produced is used in the production of chlorofluorocarbons (CFCs) and other chlorinated hydrocarbons. It was once used widely as a solvent, cleaner and degreaser, both for industrial and home use. Today, the scientific database on the effects of haloalkanes is so vast that it is no longer employed for such purposes although it is used as a model of experimental liver injury (Weber et al., 2003).  

      CCl4 is a well-known  hepato- and nephrotoxicant (Thrall et al., 2000; Ogeturket al., 2005), and proves highly useful as an experimental model for the study of certain hepatotoxic effects (Muriel et al., 2003; Moreno and Muriel, 2006). CCl4-induced toxicity, depending on dose and duration of exposure, covers a variety of effects. At low doses, transient effects prevail, such as loss of Ca2+ homeostasis, lipid peroxidation, release of noxious or beneficial cytokines (Kyung-Hyun et al., 2006; Muriel, 2007) and apoptotic events followed by regeneration. Other effects, with higher doses or longer exposure, are more serious and develop over a long period of time, such as fatty degeneration, fibrosis, cirrhosis and even cancer (Weber et al., 2003). In addition, acute intoxication with CCl4 at high doses, when the hepatocellular necrosis exceeds the regenerative capacity of the liver, fatal liver failure will ensue. Extreme doses of CCl4 result in nonspecific solvent toxicity, including central nervous system depression and respiratory failure and death. 

This study aims at investigating the effect of ethanol extract of Dennettiatripetalaon liver and kidney antioxidant enzyme activity and malondialdehyde concentration of rats exposed to CCl4.

1.1 LITERATURE REVIEW

1.1.0 THE LIVER

        The liver is the largest organ of the human body weighing approximately 1500 g, and is located in the upper right corner of the abdomen on top of the stomach, right kidney and intestines and beneath the diaphragm. The liver performs more than 500 vital metabolic functions (Naruseet al., 2007). It is involved in the synthesis of products like glucose derived from glycogenesis, plasma proteins, clotting factors and urea that are released into the bloodstream. It regulates blood levels of amino acids. 

            Liver parenchyma serves as a storage organ for several products like glycogen, fat and fat soluble vitamins. It is also involved in the production of a substance called bile that is excreted to the intestinal tract. Bile aids in the removal of toxic substances and serves as a filter that separates out harmful substances from the bloodstream and excretes them (Saukonenet al., 2006).  An excess of chemicals hinders the production of bile thus leading to the body’s inability to flush out the chemicals through waste. 

          Smooth endoplasmic reticulum of the liver is the principal ‘metabolic clearing house’ for both endogenous chemicals like cholesterol, steroid hormones, fatty acids and proteins, and exogenous substances like drugs and alcohol. The central role played by liver in the clearance and transformation of chemicals exposes it to toxic injury (Saukonenet al., 2006).  

1.1.0.1 FUNCTIONS OF THE LIVER

The liver has three main functions: storage, metabolism, and biosynthesis. Glucose is converted to glycogen and stored; when needed for energy, it is converted back to glucose. Cholesterol uptake also occurs in the liver. Fat, fat-soluble vitamins and other nutrients are also stored in the liver. Fatty acids are metabolized and converted to lipids, which are then conjugated with proteins synthesized in the liver and released into blood stream as lipoproteins. Numerous functional proteins such as, enzymes and blood-coagulating factors are also synthesized by the liver. In addition, the liver, which contains numerous xenobiotic metabolizing enzymes, is the main site of xenobiotic metabolism (Hogson and Levi, 2004).

1.1.0.2BIOTRANSFORMATION OF HEPATOTOXICANTS

Liver plays a central role in biotransformation and disposal of xenobiotics. 

The close association of liver with the small intestine and the systemic circulation enables it to maximize the processing of absorbed nutrients and minimize exposure of the body to toxins and foreign chemicals. The liver may be exposed to large concentrations of exogenous substances and their metabolites. Metabolism of exogenous compounds can modulate the properties of hepatotoxicant by either increasing its toxicity (toxication or metabolic activation) or decreasing its toxicity (detoxification).

Most of the foreign substances are lipophilic thus enabling them to cross the membranes of intestinal cells. They are rendered more hydrophilic by biochemical processes in the hepatocyte, yielding water-soluble products that are exported into plasma or bile by transport proteins located on the hepatocyte membrane and subsequently excreted by the kidney or gastrointestinal tract (Totsmannet al., 2008).

The hepatic biotransformation involves Phase I and Phase II reactions. Phase I involves oxidative, reductive, hydroxylation and demethylation pathways, primarily by way of the cytochrome P-450 enzyme system located in the endoplasmic reticulum, which is the most important family of metabolizing enzymes in the liver. The endoplasmic reticulum also contains a NADPH-dependent mixed function oxidase system, the flavin-containing monooxygenases, which oxidizes amines and sulphur compounds. 

Phase I reactions often produce toxic intermediates which are rendered non-toxic by phase II reactions. Phase II reactions involve the conjugation of chemicals with hydrophilic moieties such as glucuronide, sulfate or amino acids and lead to the formation of more water-soluble metabolite which can be excreted easily. Another Phase II reaction involves glutathione which can covalently bind to toxic intermediates by glutathione-S- transferase.  As a result, these reactions are usually considered detoxification pathways. However, this phase can also lead to the formation of unstable precursors to reactive species that can cause hepatotoxicity. 

The activities of enzymes are influenced by various endogenous factors and exogenous drugs or chemicals (Lee and Boyer, 2000). Many substances can influence the cytochrome P450 enzyme mechanism. Such substances can serve either as inhibitors or inducers. Enzyme inhibitors act immediately by blocking the metabolic activity of one or several cytochrome P450 enzymes. Enzyme inducers act slowly and increase cytochrome P450 activity by increasing its synthesis (Lynch and Price, 2007).

1.2 KIDNEY

The kidneys are bean-shaped organs that serve several essential regulatory roles in vertebrates. They remove excess organic molecules from the blood and their best known function is the removal of waste products of metabolism. They serve homeostatic functions such as the regulation of electrolytes, maintenance of acid-base balance, and regulation of blood pressure (via maintaining the salt and water balance). In producing urine, the kidneys excrete wastes such as urea and ammonium. They are responsible for the reabsorption of water, glucose, and amino acids. They also produce hormones like calcitriol and erythropoietin.

1.2.1    FUNCTIONS OF THE KIDNEY

Many of the kidney’s functions are accomplished by relatively simple mechanisms of filtration, reabsorption, and secretion, which take place in the nephron. Filtration, which takes place at the renal corpuscle, is the process by which cells and large proteins are filtered from the blood to make an ultrafiltrate that eventually becomes urine. The kidney generates 180 litres of filtrate a day, while reabsorbing a large percentage allowing for the generation of only approximately 2 litres of urine. Reabsorption is the transport of molecules from this ultrafiltrate into the blood. Secretion is the reverse process, in which molecules are transported in the opposite direction, from blood to the urine.(Bard et al., 2003).

1.2.1.0 Excretion of wastes

The kidneys excrete a variety of waste products produced by metabolism into the urine. These include the nitrogenous wastes urea, from protein catabolism, and uric acid, from nucleic acid metabolism. The ability of mammals and some birds to concentrate wastes into a volume of urine much smaller than the volume of blood from which the wastes were extracted is dependent on an elaborate countercurrent multiplication mechanism. This requires several independent nephron characteristics to operate: a tight hairpin configuration of the tubules, water and ion permeability in the descending limb of the loop, water impermeability in the ascending loop, and active ion transport out of most of the ascending limb. In addition, passive countercurrent exchange by the vessels carrying the blood supply to the nephron is essential for enabling this function.

1.2.1.1 Reabsorption of the vital nutrients

Glucose at normal plasma levels is completely reabsorbed in the proximal tubule. The mechanism for this is the Na+/glucose cotransporter. A plasma level of 350mg/dL will fully saturate the transporters and glucose will be lost in the urine. A plasma glucose level of approximately 160 is sufficient to allow glucosuria, which is an important clinical clue to diabetes mellitus.

Amino acids are reabsorbed by sodium dependent transporters in the proximal tubule. Hartnup disease is a deficiency of the tryptophan amino acid transporter which results in pellagra (Le Tao, 2013)

1.2.1.2 Acid-base homeostasis

Two organ systems, the kidneys and lungs, maintain acid base homeostasis, which is the maintenance of pH around a relatively stable value. The lungs contribute to acid-base homeostasis by regulating carbon dioxide (CO2) concentration. The kidneys have two very important roles in maintaining the acid-base balance: to reabsorb and regenerate bicarbonate from urine, and to excrete hydrogen ions and fixed acids (anions of acids) into urine (Seldinet al., 1989).

1.2.1.3 Osmolality regulation

 Any significant rise in plasma osmolality is detected by the hypothalamus, which communicates directly with the posterior pituitary gland. An increase in osmolality causes the gland to secrete antidiuretic hormone (ADH), resulting in water reabsorption by the kidney and an increase in urine concentration. The two factors work together to return the plasma osmolality to its normal levels.

ADH binds to principal cells in the collecting duct that translocate aquaporins to the membrane, allowing water to leave the normally impermeable membrane and be reabsorbed into the body by the vasa recta, thus increasing the plasma volume of the body.

There are two systems that create a hyperosmotic medulla and thus increase the body plasma volume: urea recycling and the ‘single effect’.

Urea is usually excreted as a waste product from the kidneys. However, when plasma blood volume is low and ADH is released the aquaporinsthat are opened are also permeable to urea. This allows urea to leave the collecting duct into the medulla creating a hyperosmotic solution that attracts water. Urea can then re-enter the nephron and be excreted or recycled again depending on whether ADH is still present or not. The ‘single effect’ describes the fact that the ascending thick limb of the loop of henle is not permeable to water but is permeable to NaCl. This allows for a countercurrent exchange system whereby the medulla becomes increasingly concentrated, but at the same time setting up an osmotic gradient for water to follow should the aquaporins of the collecting duct be opened by ADH (Vander, 1985).

1.2.1.4 Blood pressure regulation

Although the kidney cannot directly sense blood, long term regulation of blood pressure predominantly depends upon the kidney. This primarily occurs through maintenance of the extracellular fluid compartment, the size of which depends on the plasma sodium concentration. Renin is the first in the series of important chemical messengers that make up the renin-angiotensin system. Changes in rennin ultimately alter the output of this system, principally the hormones angotensin II and aldostrone. Each hormone acts via multiple mechanisms, but both increase the kidney’s absorption of sodium chloride, thereby expanding the extracellular fluid compartment, and an increase in blood pressure. Conversely, when rennin levels are low, angiotensin II and aldosterone levels decrease, contracting the extracellular fluid compartment, and an increase in blood pressure. Conversely, when rennin levels are low, angiotensin II and aldosterone levels decrease, contracting the extracellular fluid compartment, and decreasing blood pressure.

1.2.1.5 Hormone secretion

The kidneys secrete a variety of hormones, including erythropoietin, and the enzyme rennin. Erythropoietin is released in response to hypoxia (low levels of oxygen at tissue level) in the renal circulation. It stimulates erythropoiesis (production of red blood cells) in the bone marrow. Calcitriol, the activated form of vitamin D, promotes intestinal absorption of calcium and the renal reabsorption of phosphate. Part of the renin-angiotensin-aldosterone system, renin is an enzyme involved in the regulation of aldosterone levels (Valtin, 1983).

1.3 HEPATOTOXICITY

         Hepatotoxicity refers to liver dysfunction or liver damage that is associated with an overload of drugs or xenobiotics(Navaroet al., 2006). The chemicals that cause liver injury are called hepatotoxins or hepatotoxicants. Hepatotoxicants are exogenous compounds of clinical relevance and may include overdoses of certain medicinal drugs, industrial chemicals, natural chemicals like microcystins, herbal remedies and dietary supplements (Willett et al., 2004). 

         Certain drugs may cause liver injury when introduced even within the therapeutic ranges. Hepatotoxicity may result not only from direct toxicity of the primary compound but also from a reactive metabolite or from an immunologically-mediated response affecting hepatocytes, biliary epithelial cells and/or liver vasculature (Saukkonenet al., 2006).

           The hepatotoxic response elicited by a chemical agent depends on the concentration of the toxicant which may be either parent compound or toxic metabolite, differential expression of enzymes and concentration gradient of cofactors in blood across the acinus.  Hepatotoxic response is expressed in the form of characteristic patterns of cytolethality in specific zones of the acinus.

1.3.1 SYMPTOMS OF HEPATOTOXICITY

           Hepatotoxicity related symptoms may include a jaundice or icterus appearance causing yellowing of the skin, eyes and mucous membranes due to high level of bilirubin in the extracellular fluid, pruritus, 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 (Bleibelet al., 2007; Chang and Shaino, 2007).

          The symptoms of hepatotoxicity can be subdivided into clinical and drug-induced pathological symptoms.

1.3.1.0 CLINICAL MANIFESTATION

      The manifestation of drug induced hepatotoxicity is highly variable, ranging from a symptomatic evaluation of liver enzymes to fulminant hepatic failure. The injury may suggest a hepatocellular injury with evaluation of aminotransferases levels as the predominant symptom or a cholestatic injury, with elevated alkaline phosphatase levels with or without hyperbiliruminemia being the main feature.

       In addition, drugs that cause mild amino transferase elevation with subsequent adaptation are differentiated from those that result in true toxicity that require discontinuation.

Hepatotoxicity can be induced in the laboratory by exposing laboratory animals to toxic chemicals such as carbon tetrachloride.

1.3.1.1 PATHOLOGICAL MANIFESTATION

Acute hepatocellular damage

Chronic hepatocellular damage

Chronic cholestasis

Vascular lesions / venocclusive disease

Angiosarcoma (Bleibelet al., 2007; Chang and Shaino, 2007).

1.4 CARBON TETRACHLORIDE 

Carbon tetrachloride (CCl4) is an industrial chemical that does not occur naturally. Most of the carbon tetrachloride produced is used in the production of chlorofluorocarbons (CFCs) and other chlorinated hydrocarbons. It was once used widely as a solvent, cleaner and degreaser, both for industrial and home use. Today, the scientific database on the effects of haloalkanes is so vast that it is no longer employed for such purposes although it is used as a model of experimental liver injury (Weber et al., 2003).  

      CCl4 is a well-known hepato- and nephrotoxicant (Thrall et al., 2000; Ogeturket al., 2005), and proves highly useful as an experimental model for the study of certain hepatotoxic effects (Muriel et al., 2003; Moreno and Muriel, 2006). CCl4-induced toxicity, depending on dose and duration of exposure, covers a variety of effects. At low doses, transient effects prevail, such as loss of Ca2+ homeostasis, lipid peroxidation, release of noxious or beneficial cytokines (Kyung-Hyun et al., 2006; Muriel, 2007) and apoptotic events followed by regeneration. Other effects, with higher doses or longer exposure, are more serious and develop over a long period of time, such as fatty degeneration, fibrosis, cirrhosis and even cancer (Weber et al., 2003). In addition, acute intoxication with CCl4 at high doses, when the hepatocellular necrosis exceeds the regenerative capacity of the liver, fatal liver failure will ensue. Extreme doses of CCl4 result in nonspecific solvent toxicity, including central nervous system depression and respiratory failure and death. CCl4 can be administered orally, intravenously and intraperitonially.

           CCl4 metabolism begins with the formation of the trichloromethyl free radical, CCl3 through the action of the mixed function cytochrome P-450 oxygenase system of the endoplasmic reticulum. This process involves reductive cleavage of a carbon-chlorine bond. Free radical activation of CCl4 in mitochondria has also been observed and may contribute significantly to its toxicity. The major cytochrome iso-enzyme to execute biotransformation of CCl4 is cytochrome P-450 iso-enzyme 2E1 (CYP2E1). This is evidenced by the absence of toxicity in CYP2E1 knockout mice. 

          In humans, CYP2E1 dominates CCl4 metabolism at environmentally relevant concentrations, but at higher concentrations other cytochromes, particularly CYP3A, also contribute importantly (Zangeret al., 2000).  The CCl3 radical reacts with several important biological substances, like fatty acids, proteins, lipids, nucleic acids and amino acids (Weber et al., 2003).  CCl3 also acts by abstracting hydrogen from unsaturated fatty acids to form chloroform. DNA

adducts is a mechanism for CCl4-induced carcinogenesis.Image

(FIG. 1.4)A schematic diagram explaining the onset of steatosis involving MTP degradation of CCl4.  (Panet al., 2007)CCl4 is converted to free radicals (CCl3and Cl) by cytochrome P450 oxygenases. MTP is covalently modified by CCl3, ubiquitinylated, and degraded by proteasomes. This leads to increased accumulation of triglycerides and cholesterol in the tissues. If proteasomal degradation of MTP is inhibited by lactacystin, the CCl4 toxicity is averted in part because the protected MTP is able to assist in lipoprotein assembly and in the secretion of triglycerides.

1.5 OXIDATIVE STRESS

       Oxidative stress occurs when the balance between antioxidants and ROS are disrupted because of either depletion of antioxidants or accumulation of ROS. When oxidative stress occurs, cells attempt to counteract the oxidant effects and restore the redox balance by activation or silencing of genes encoding defensive enzymes, transcription factors, and structural proteins (Scandalioset al., 2004). Ratio between oxidized and reduced glutathione (2GSH/GSSG) is one of the important determinants of oxidative stress in the body. Higher production of ROS in body may change DNA structure, result in modification of proteins and lipids, activation of several stress-induced transcription factors, and production of pro-inflammatory and anti-inflammatory cytokines.

1.5.1 EFFECTS OF OXIDATIVE STRESS ON DNA 

ROS can lead to DNA modifications in several ways, which involves degradation of bases, single- or double- stranded DNA breaks, purine, pyrimidine or sugar-bound modifications, mutations, deletions or translocations, and cross-linking with proteins. Most of these DNA modifications are highly relevant to carcinogenesis, aging, and neurodegenerative, cardiovascular, and autoimmune diseases. Tobacco smoke, redox metals, and non-redox metals, such as iron, cadmium, chrome, and arsenic, are also involved in carcinogenesis and aging by generating free radicals or binding with thiol groups. Formation of 8-OH-G is the best- known DNA damage occurring via oxidative stress and is a potential biomarker for carcinogenesis. Promoter regions of genes contain consensus sequences for transcription factors. These transcription factor–binding sites contain GC-rich sequences that are susceptible for oxidant attacks. Formation of 8-OH-G DNA in transcription factor binding sites can modify binding of transcription 

factors and thus change the expression of related genes as has been shown for AP-1 and Sp-1 target sequences. Besides 8-OH-G, 8,59-cyclo-29-deoxyadenosine (cyclo-dA) has also been shown to inhibit transcription from a reporter gene in a cell system if located in a TATA box (Marietta et al., 2002).The TATA-binding protein initiates transcription by changing the bending of DNA.

Oxidative stress causes instability of microsatellite (short tandem repeats) regions. Redox active metal ions, hydroxyl radicals increase microsatellite instability.Even though single-stranded DNA breaks caused by oxidant injury can easily be tolerated by cells, double-stranded DNA breaks induced by ionizing radiation can be a significant threat for the cell survival (Caldecott et al., 2003). Methylation at CpG islands in DNA is an important epigenetic mechanism that may result in gene silencing. Oxidation of 5-MeCyt to 5-hydroxymethyl uracil (5-OHMeUra) can occur via deamination/oxidation reactions of thymine or 5-hydroxymethyl cytosine intermediates (Cooke et al., 2003).  In addition to the modulating gene expression, DNA methylation also seems to affect chromatin organization. Aberrant DNA methylation patterns induced by oxidative attacks also affect DNA repair activity.

1.5.2 EFFECTS OF OXIDATIVE STRESS ON LIPIDS 

ROS can induce lipid peroxidation and disrupt the membrane lipid bilayer arrangement that may inactivate membrane-bound receptors and enzymes and increase tissue permeability.Products of lipid peroxidation, such as MDA and unsaturated aldehydes, are capable of inactivating many cellular proteins by forming protein cross-linkages. 4-Hydroxy-2-nonenal causes depletion of intracellular GSH and induces of peroxide production,activates epidermal growth factor receptor,and induces fibronectin production (Tsukagoshiet al., 2002).  Lipid peroxidation products, such as isoprostanes and thiobarbituric acid reactive substances, have been used as indirect biomarkers of oxidative stress, and increased levels were shown in the exhaled breath condensate or broncho-alveolar lavage fluid or lung of chronic obstructive pulmonary disease patients or smokers (Montuschiet al., 2000).

1.5.3 EFFECTS OF OXIDATIVE STRESS ON PROTEINS 

         ROS can cause fragmentation of the peptide chain, alteration of electrical charge of proteins, cross-linking of proteins, and oxidation of specific amino acids and therefore lead to increased susceptibility to proteolysis by degradation by specific proteases (Kelly et al., 2003). Cysteine and methionine residues in proteins are particularly more susceptible to oxidation.  Oxidation of sulfhydryl groups or methionine residues of proteins cause conformational changes, protein unfolding, and degradation.  Enzymes that have metals on or close to their active sites are especially more sensitive to metal catalyzed oxidation. Oxidative modification of enzymes has been shown to inhibit their activities. In some cases, specific oxidation of proteins may take place. For example, methionine can be oxidized methionine sulfoxide and phenylalanine to o-tyrosine sulfhydryl groups can be oxidized to form disulfide bonds and carbonyl groups may be introduced into the side chains of proteins. Gamma rays, metal-catalyzed oxidation, HOCl, and ozone can cause formation of carbonyl groups (Shacteret al., 2000).

1.5.4 LIPID PEROXIDATION

        Currently, lipid peroxidation is considered as one the main molecular mechanisms involved in the oxidative damage to cell structures and in the toxicity process that lead to cell death. First, lipid peroxidation was studied for food scientists as a mechanism for the damage to alimentary oils and fats, nevertheless other researchers considered that lipid peroxidation was the consequence of toxic metabolites (e.g. CCl4) that produced highly reactive species, disruption of the intracellular membranes and cellular damage (Dianzani and Barrera, 2008).

       Lipid peroxidation is a complex process known to occur in both plants and animals. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids and the eventual destruction of membrane lipids, with the production of a variety of breakdown products, including alcohols, ketones, alkanes, aldehydes and ethers (Dianzani and Barrera, 2008). In pathological situations the reactive oxygen and nitrogen species are generated at higher than normal rates, and as a consequence, lipid peroxidation occurs with α -tocopherol deficiency. In addition to containing high concentrations of polyunsaturated fatty acids and transition metals, biological membranes of cells and organelles are constantly being subjected to various types of damage. The mechanism of biological damage and the toxicity of these reactive species on biological systems are currently explained by the sequential stages of reversible oxidative stress and irreversible oxidative damage. The lipid peroxidation reaction involves three major steps:

1. Initiation step

2. Propagation step

3. Termination step

Image

Mechanism of lipid peroxidation (Wang, 1999)

INITIATION

Initiation is the step whereby a fatty acid radical is produced. The initiators in living cells and most notably reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl groups as illustrated in the peroxidation mechanism. This reactive oxygen species combine with a hydrogen atom in the liquid molecule to make water and a fatty acid radical (Kanner, 1987).

PROPAGATION

     The fatty acid radicals are not very stable molecules and so, they react readily with molecular oxygen, thereby creating peroxyl fatty acid radicals. This radical is an unstable species that react with another free fatty acid producing a different fatty acid radical and lipid peroxide or cyclic peroxide if it had reacted with itself. The cycle continues as the new fatty acid radicals react in the same way (Marnett, 1999).

TERMINATION

    When a radical reacts, it produces another radical this is why the process is called a “chain reaction mechanism”. The radical reaction stops when two radicals react and produce a non- radical species. This happens only when the concentration of radical species is high enough for there to be a high probability of two radicals actually colliding. Living organisms have evolved different molecules that speed up termination by catching free radicals and therefore protect cell membrane. 

1.6 ANTIOXIDANTS

       The human body is equipped with a variety of antioxidants that serve to counterbalance the effect of oxidants. For all practical purposes, these can be divided into 2 categories: enzymatic and non-enzymatic. 

1.6.0 ENZYMATIC ANTIOXIDANTS

           The major enzymatic antioxidants of the lungs are SODs, catalase, and GSH-Px. In addition to these major enzymes, other antioxidants, including heme oxygenase-1, and redox proteins, such as thioredoxins (TRXs), peroxiredoxins (PRXs) and glutaredoxins, have also been found to play crucial roles in the pulmonary antioxidant defenses. 

1.6.0.1 SUPEROXIDE DISMUTASE

Since superoxide is the primary ROS produced from a variety of sources, its dismutation by SOD is of primary importance for each cell. All three forms of SOD, that is, CuZn- SOD, Mn-SOD, and EC-SOD, are widely expressed in the human lung. Mn-SOD is localized in the mitochondria matrix. EC-SOD is primarily localized in the extracellular matrix, especially in areas containing high amounts of type I collagen fibers and around pulmonary and systemic vessels. It has also been detected in the bronchial epithelium, alveolar epithelium, and alveolar macrophages (Kinnulaet al., 2003)

Overall, CuZn- SOD and Mn-SOD are generally thought to act as bulk scavengers of superoxide radicals. The relatively high EC-SOD level in the lung with its specific binding to the extracellular matrix components may represent a fundamental component of lung matrix protection (Zelkoet al., 2003).

1.6.0.2 CATALASE

H2 O2 that is produced by the action of SODs or the action of oxidases, such as xanthine oxidase, is reduced to water by catalase and the GSH-Px. Catalase exists as a tetramer composed of 4 identical monomers, each of which contains a heme group at the active site. Degradation of H2 O2 is accomplished via the conversion between 2 conformations of catalase-ferricatalase (iron coordinated to water) and compound I (iron complexed with an oxygen atom). Catalase also binds NADPH as a reducing equivalent to prevent oxidative inactivation of the enzyme (formation of compound II) by H2 O2 as it is reduced to water.69 Enzymes in the redox cycle responsible for the reduction of H2 O2 and lipid hydroperoxides (generated as a result of membrane lipid peroxidation) include the GSH-Pxs. 

1.6.0.3 GLUTATHIONE PEROXIDASE (GSH-Px)

The GSH-Pxs are a family of tetrameric enzymes that contain the unique amino acid selenocysteine within the active sites and use low-molecular-weight thiols, such as GSH, to reduce H2 O2 and lipid peroxides to their corresponding alcohols. Four GSH- Pxs have been described, encoded by different genes: GSH- Px-1 (cellular GSH-Px) is ubiquitous and reduces H2 O2 and fatty acid peroxides, but not esterified peroxyl lipids (Arthur et al., 2000) Esterified lipids are reduced by membrane-bound GSH-Px-4 (phospholipid hydroperoxide GSH-Px), which can use several different low-molecular-weight thiols as reducing equivalents. GSH-Px-2 (gastrointestinal GSH-Px) is localized in gastrointestinal epithelial cells where it serves to reduce dietary peroxides.  GSH-Px-3 (extracellular GSH-Px) is the only member of the GSH-Px family that resides in the extracellular compartment and is believed to be one of the most important extracellular antioxidant enzyme in mammals. Of these, extracellular GSH-Px is most widely investigated in the human lung.  In addition, disposal of H2 O2 is closely associated with several thiol-containing enzymes, namely, TRXs (TRX1 and TRX2), thioredoxinreductases (TRRs), PRXs (which are thioredoxin peroxidases), and glutaredoxins (Gromeret al., 2004).

Two TRXs and TRRs have been characterized in human cells, existing in both cytosol and mitochondria. In the lung, TRX and TRR are expressed in bronchial and alveolar epithelium and macrophages. Six different PRXs have been found in human cells, differing in their ultra-structural compartmentalization. Experimental studies have revealed the importance of PRX VI in the protection of alveolar epithelium. Human lung expresses all PRXs in bronchial epithelium, alveolar epithelium, and macrophages (Kinnulaet al., 2002).  PRX V has recently been found to function as a peroxy-nitrite reductase, which means that it may function as a potential protective compound in the development of ROS-mediated lung injury (Holmgren et al., 2000). Common to these antioxidants is the requirement of NADPH as a reducing equivalent. NADPH maintains catalase in the active form and is used as a cofactor by TRX and GSH reductase, which converts GSSG to GSH, a co-substrate for the GSH-Pxs. Intracellular NADPH, in turn, is generated by the reduction of NADP1 by glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the pentose phosphate pathway, during the conversion of glucose- 6-phosphate to 6-phosphogluconolactone. By generating NADPH, glucose-6-phosphate dehydrogenase is a critical determinant of cytosolic GSH buffering capacity (GSH/ GSSG) and therefore, can be considered an essential, regulatory antioxidant enzyme (Dickinson et al., 2002). GSTs, another antioxidant enzyme family, inactivate secondary metabolites, such as unsaturated aldehydes, epoxides, and hydroperoxides. Three major families of GSTs have been described: cytosolic GST, mitochondrial GST, and membrane-associated microsomal GST that has a role in eicosanoid and GSH metabolism. 

1.6.1 NON-ENZYMATIC ANTIOXIDANTS

Non-enzymatic antioxidants include low-molecular-weight compounds, such as vitamins (vitamins C and E), b-carotene, uric acid, and GSH, a tripeptide (L-g-glutamyl-L-cysteinyl-L- glycine) that comprise a thiol (sulfhydryl) group.

1.6.1.1 Vitamin C (Ascorbic Acid)

Water-soluble vitamin C (ascorbic acid) provides intracellular and extracellular aqueous-phase antioxidant capacity primarily by scavenging oxygen free radicals. It converts vitamin E free radicals back to vitamin E. Its plasma levels have been shown to decrease with age.

1.6.1.2 Vitamin E (α-Tocopherol)

         Lipid-soluble vitamin E is concentrated in the hydro- phobic interior site of cell membrane and is the principal defense against oxidant-induced membrane injury. Vitamin E donates electron to peroxyl radical, which is produced during lipid peroxidation. α-Tocopherol is the most active form of vitamin E and the major membrane-bound antioxidant in cell. Vitamin E triggers apoptosis of cancer cells and inhibits free radical formations.

1.6.1.3 Glutathione

GSH is highly abundant in all cell compartments and is the major soluble antioxidant. GSH/GSSG ratio is a major determinant of oxidative stress. GSH shows its antioxidant effects in several ways. It detoxifies hydrogen peroxide and lipid peroxides via action of GSH-Px. GSH donates its electron to H2 O2 to reduce it into H2 O and O2. GSSG is again reduced into GSH by GSH reductase that uses NAD(P)H as the electron donor. GSH-Pxs are also important for the protection of cell membrane from lipid peroxidation. Reduced glutathione donates protons to membrane lipids and protects them from oxidant attacks. GSH is a cofactor for several detoxifying enzymes, such as GSH-Px and transferase. It has a role in converting vitamin C and E back to their active forms. GSH protects cells against apoptosis by interacting with pro-apoptotic and anti-apoptotic signaling pathways (Masellaet al., 2005). 

1.7 LIVER FUNCTION TESTS

         When the liver is damaged, it cannot carry out its functions effectively. Certain enzymes and metabolites can provide a basis for the test of liver functionality.  Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), Ƴ-glutamyltranspeptidase (GGT), are among a class of enzymes called liver marker enzymes. Although not entirely specific to the liver, these enzymes along with molecules like albumin and bilirubin are used as markers for liver function.

1.7.1 ALANINE AMINOTRANSFERASE (ALT)

Perhaps the most commonly used indicator of liver (hepatocellular) damage are the ALT and AST, formerly called SGPT and SGOT respectively. These enzymes are normally found in liver cells, but a dysfunction or injury could lead to a leakage of these enzymes into the blood. The ALT is thought to be a more specific indicator of liver inflammation as AST is also found in other organs such as the heart and skeletal muscle. In acute injury to the liver, as in viral hepatitis, the level of ALT and AST may be used as a general measure of the degree of liver inflammation or damage. The higher the ALT level, the more cell death or inflammation of the liver enzyme occurred (Dial, 1995).

1.7.2 ASPARTATE AMINOTRANSFERASE (AST)

AST is the enzyme that is produced by both the liver and the muscle. The level of AST also increases in cases of heart attack. In many cases of liver inflammation, the ALT and AST levels are equally elevated. In some conditions such as hepatitis, AST levels may be higher than ALT level. AST levels can be normal yet there can be liver damage occurring. This test adds one more perspective to the picture of liver disease (Dial, 1995).

1.7.3 ALKALINE PHOSPHATASE

It is produced in the bile duct, kidney placement, and bones. This enzyme is measured or determined to help the physicians to know if the disease is concentrated in the bile duct or in the liver. When this enzyme level is high, and the level of ALT and AST are fairly normal, problems associated with the bile duct may be determined (Wright, 1993).

1.7.4 BILIRUBIN

Bilirubin is the main bile pigment in humans which, when elevated causes the yellow discoloration of the skin called jaundice. It is a reddish yellow pigment formed by the breakdown of hemoglobin in worn out red blood cells. The liver helps to conjugate bilirubin with glucuronic acid, forming bilirubin di-glucuronide (its conjugated form). The level of bilirubin in the blood can be elevated due to over production, decreased uptake by the liver, decreased conjugation, decreased secretion from the liver or blockage of the bile duct. In cases of increased production, decreased secretion from the liver or bile duct obstruction, de-conjugated or indirect bilirubin will be primarily elevated. Many different liver diseases can cause elevated bilirubin levels (Crook, 2006).

1.7.5 ALBUMIN

Albumin is produced entirely in the liver and constitutes about 60% of total serum protein. It is important in regulating the flow of water between the plasma and tissue fluid by its effect on plasma colloid osmotic pressure (oncotic pressure). When the concentration of albumin is significantly reduced, the plasma osmotic pressure is insufficient to draw water from the tissue spaces back into the plasma. This leads to a build-up of fluids within the tissue spaces, referred to as oedema.

1.8 LIPID PROFILE

1.8.1 TOTAL CHOLESTEROL

Cholesterol is a waxy fat like substance that is important for normal body functioning. Cholesterol is used for cellular functions and for the production of hormones. Your body, in most cases will produce enough cholesterol to maintain normal body needs. The liver is the major production factory for cholesterol (about 70%). Diets high in saturated fats significantly increase the amount of cholesterol in the blood stream. Research indicates that diets high in saturated and total fat play a significant role in the process of atherosclerosis (plaque build-up on the artery walls). A cholesterol value of 220mg/dl correlates to nearly two-fold elevation in incidence of coronary heart disease as compared to 180mg/dl (Tymoczkoet al., 2010).

1.8.2 TRIGLYCERIDES

It is an ester derived from glycerol and 3 fatty acids. It is a main constituent of vegetable oil and animal fats (Jonkeret al., 2003). Triglycerides are a major component of very low density lipoproteins and chylomicrons, and they play a role in metabolism as energy source and transporters of dietary fats.  High level of triglycerides in the blood stream has been linked to atherosclerosis and by extension, the risk of heart disease and stroke (Jonker et al., 2002).

1.9 DENNETIA TRIPETALA

Dennettiatripetalaalso known as pepper fruit tree is a well-known Nigerian spicy medicinal plant.  It is found in the tropical rainforest region of Nigeria and sometimes in Savanna areas (Okwu et al., 2005).  It is locally called “Nkarika” by the Efiks of Calabar.  The young leaves and fruits have dinstinctive spicy taste.  The mature fruits constitute the main edible portions.  Some communities in parts of Southern Nigeria also utilize the leaves and roots, in addition to the fruits for medicinal purpose.  Dennettiatripetala is used as masticators, which when chewed produces unique peppery effect. The peppery spicy taste of mature Dennettiatripetala fruits usually serves as a mild stimulant to the consumer.

1.9.1 METHOD OF PROPAGATION

Dennettiatripetala is propagated by direct seedling and use of root suckers. Dennettiatripetala is also propagated by natural regeneration.

1.9.2 ETHNOMEDICINAL USES OF DENNETIA TRIPETALA

Dennettiatripetala is commonly known as pepper fruit by the English, “mmimi” by the Igbos, “Nkaika” by the Ibibio and Efik, “Imako” by the Urhobo tribe of the Niger-Delta region, and “Igberi” by the Yorubas.).Dennettiatripetala (pepper fruit) is a medium sized tree found commonly in the tropical rainforest region of Nigeria and sometimes in savannah areas. Dennettiatripetala, or pepper fruit tree, is a well-known Nigerian spicy medicinal plant. It is an indigenous medium sized or small woody shrub. It is commonly found in the rain-forest and occasionally in the savanna. (Timothy and Okere, 2008).The tree grows up to 12m-15m in height and 0.6m in girth, with a dense compact crown. The wood is soft, white coloured and prone to termite attack. It has a fibrous bark which has a strong scent. The leaves are 3 – 6 inches long by 1.5–2.5 inches broad, elliptic to ovate, shortly acuminate broadly connate to rounded at the base. The flowers are light brown outside, reddish inside and usually in small clusters on the young or older wood. The fruits are green at first but eventually turn reddish pink when ripe with finger-like carpel constricted between the seeds. The fruits are edible and rich in vitamin C , both the fruits and young leaves have a distinctive spicy taste, the bark of  Dennettiatripetala fruits is mixed with food to create variation in the taste and flavour of different foods.(Okafor, 1980).It has been reported that the peppery fruits of Dennettiatripetala usually find application in food meant for pregnant women. Dennettiatripetala fruit contains several nutrients and biologically active components that prolong and enhance life. It is a good source of ascorbic acid, riboflavin, thiamine and niacin. Therefore, this fruit is nutritionally necessary for a well-balanced diet because it contributes important vitamins such as vitamin C (ascorbic acid) which can be used for the treatment of the common cold and the control of other diseases such as prostate cancer.  The bark of the tree is mixed with food to create variation in the taste and flavour of different food .The leaves are used in folk medicine for the treatment of fever, cough, asthma, catarrh, toothache, diarrhoea and rheumatism the fruit reduces the risk of blindness caused by glaucoma.

1.9.3 BOTANICAL DESCRIPTION OF DENNETIA TRIPETALA

Image

Fig 1 Fruits of  Dennettia. tripetala

Dennettiatripetala (pepper fruit) is an indigenous fruit tree of the family Annonaceae  (Etukudo, 2000). It is a medium- sized or small tree which spreads throughout the rain forest and sometimes found in forest within the Savanna areas matured fruits constitute the main edible portion. The leaves, fruit, bark and root of the plants possess strong peppery and pungent spicy taste with a characteristic aroma and fragrance. The young leaves and fruits have instinctive spicy taste. The fruits are chewed in different forms (fresh green, fresh ripened red, black dry fruit and dry seed). The bark is smooth to roughly scaly, grey to brown, often with some distinct purple layers. 

1.9.4 COMMERCIAL / ECONOMIC IMPORTANCE OF DENNETTIATRIPETALA

The parts used include the leaves, fruits, seeds, roots and stem. A survey of existing literatures shows that pepper fruits contain essential oils and phenolic acids, ethanol, alkaloid, ethyl acetate, flavonoids, tannins and glycosides. Indications show that the rich presence of a type of Dennettiaessential oil called oleoresins determines the aromatic flavouring, colouring and pungent properties of Dennettiatripetala. Medicinally, the leaves and fruits are used for cough treatment and enhancing appetite. In Igbo land, the fruits and seed are signs of hospitality for visitors. The wood is white and soft which yields a good fuel wood.  The fruit tree is a tropical tree common in the mangrove forests of the west coast of Africa. The fruit of Dennettiatripetala is quite popular in Southern Nigeria where it serves for cultural entertainment of guests, particularly during coronation, the new yam festival and marriage ceremonies.

1.9.5 NUTRITIONAL EVALUATION OF UNRIPE AND RIPE PEPPER FRUIT (DENNETTIA TRIPETALA)

The nutritional evaluation of unripe Dennettiatripetalain percentage wet basis (Table 1) revealed protein (6.59%), moisture content (15.26%), fat (5.52%), ash (4.13%), fiber (17.05%) and carbohydrate (51.45). Its mineral content comprises of calcium (181.69mg/g), magnesium (229.78mg/g), iron (0.2mg/g), phosphorus (285.8mg/g), potassium (360.8mg/g) and Sodium (6.12m/mg). The vitamins include ascorbic acid (85.65mg/g), niacin (0.40mg/g), thiamine (0.10mg/g), riboflavin (0.05mg/g) and vitamin A (65.58mg/g) while ripe Dennettiatripetalashowed protein (4.67%), moisture content (18.73%), fat (5.78%), Ash (3.18%), fiber(14.32%) and carbohydrate (53.32%).Evaluation of the ripe pepper fruit for its mineral content indicated calcium (138.94mg/g),magnesium (173.68mg/g), iron (0.23mg/g), phosphorus (243.8mg/g), potassium(324.27mg/g) and sodium (5.47mg/g). The study also revealed ascorbic acid (115.57mg/g),niacin (0.37mg/g), thiamine (0.08mg/g), riboflavin (0.05mg/g) and vitamin A (388.10mg/g).(Okwu and Morah, 2004).

Table 1. Proximate composition DennettiaTripetala (Pepper fruit)

% Samples

Nutrient                        Unripe             Ripe

Moisture content       15.26 ± 0.07      18.73 ± 0.02

Protein                         6.59 ± 0.08        4.67 ± 0.08

Fat                                5.52 ± 0.3          5.78 ± 0.08

Ash                               4.13 ± 0.02        3.18 ± 0.03

Fiber                          17.05 ± 0.7        14.32 ± 0.3

Carbohydrate             51.45 ± 0.015    53.32 ± 0.02

(Values are means of triplicate analysis)

(Ihemejeet al., 2013)

The unripe pepper fruit (Dennettiatripetala) showed higher value in protein (6.59%), ash (4.13%) and fiber (17.05%) than the ripe Dennettiatripetalawhich had protein (4.67%), ash (3.18%) and fiber (14.32%). Ripe Dennettiatripetalashowed high moisture content (18.73%), fat (5.78%) and carbohydrate (53.32%) than the unripe. Increased moisture content in a food material increases the chances of microbial attack. This justifies why ripe Dennettiatripetalais more prone to microbial spoilage than the unripe. The high ash content of unripe pepper fruit is a reflective of its greater mineral content than the ripe (Table 2) which makes it important for little children. Minerals enhance the important functions of maintaining

.

EFFECT OF ETHANOL EXTRACT OF DENNETTIATRIPETALA ON LIVER AND KIDNEY ANTIOXIDANT ENZYME ACTIVITY AND MALONDIALDEHYDE CONCENTRATION OF ALBINO WISTAR RATS EXPOSED TO CCL4.



TYPE IN YOUR TOPIC AND CLICK SEARCH.






RESEARCHWAP.ORG
Researchwap.org is an online repository for free project topics and research materials, articles and custom writing of research works. We’re an online resource centre that provides a vast database for students to access numerous research project topics and materials. Researchwap.org guides and assist Postgraduate, Undergraduate and Final Year Students with well researched and quality project topics, topic ideas, research guides and project materials. We’re reliable and trustworthy, and we really understand what is called “time factor”, that is why we’ve simplified the process so that students can get their research projects ready on time. Our platform provides more educational services, such as hiring a writer, research analysis, and software for computer science research and we also seriously adhere to a timely delivery.

TESTIMONIES FROM OUR CLIENTS


Please feel free to carefully review some written and captured responses from our satisfied clients.

  • "Exceptionally outstanding. Highly recommend for all who wish to have effective and excellent project defence. Easily Accessable, Affordable, Effective and effective."

    Debby Henry George, Massachusetts Institute of Technology (MIT), Cambridge, USA.
  • "I saw this website on facebook page and I did not even bother since I was in a hurry to complete my project. But I am totally amazed that when I visited the website and saw the topic I was looking for and I decided to give a try and now I have received it within an hour after ordering the material. Am grateful guys!"

    Hilary Yusuf, United States International University Africa, Nairobi, Kenya.
  • "Researchwap.org is a website I recommend to all student and researchers within and outside the country. The web owners are doing great job and I appreciate them for that. Once again, thank you very much "researchwap.org" and God bless you and your business! ."

    Debby Henry George, Massachusetts Institute of Technology (MIT), Cambridge, USA.
  • "Great User Experience, Nice flows and Superb functionalities.The app is indeed a great tech innovation for greasing the wheels of final year, research and other pedagogical related project works. A trial would definitely convince you."

    Lamilare Valentine, Kwame Nkrumah University, Kumasi, Ghana.
  • "I love what you guys are doing, your material guided me well through my research. Thank you for helping me achieve academic success."

    Sampson, University of Nigeria, Nsukka.
  • "researchwap.com is God-sent! I got good grades in my seminar and project with the help of your service, thank you soooooo much."

    Cynthia, Akwa Ibom State University .
  • "Sorry, it was in my spam folder all along, I should have looked it up properly first. Please keep up the good work, your team is quite commited. Am grateful...I will certainly refer my friends too."

    Elizabeth, Obafemi Awolowo University
  • "Am happy the defense went well, thanks to your articles. I may not be able to express how grateful I am for all your assistance, but on my honour, I owe you guys a good number of referrals. Thank you once again."

    Ali Olanrewaju, Lagos State University.
  • "My Dear Researchwap, initially I never believed one can actually do honest business transactions with Nigerians online until i stumbled into your website. You have broken a new legacy of record as far as am concerned. Keep up the good work!"

    Willie Ekereobong, University of Port Harcourt.
  • "WOW, SO IT'S TRUE??!! I can't believe I got this quality work for just 3k...I thought it was scam ooo. I wouldn't mind if it goes for over 5k, its worth it. Thank you!"

    Theressa, Igbinedion University.
  • "I did not see my project topic on your website so I decided to call your customer care number, the attention I got was epic! I got help from the beginning to the end of my project in just 3 days, they even taught me how to defend my project and I got a 'B' at the end. Thank you so much researchwap.com, infact, I owe my graduating well today to you guys...."

    Joseph, Abia state Polytechnic.
  • "My friend told me about ResearchWap website, I doubted her until I saw her receive her full project in less than 15 miniutes, I tried mine too and got it same, right now, am telling everyone in my school about researchwap.com, no one has to suffer any more writing their project. Thank you for making life easy for me and my fellow students... Keep up the good work"

    Christiana, Landmark University .
  • "I wish I knew you guys when I wrote my first degree project, it took so much time and effort then. Now, with just a click of a button, I got my complete project in less than 15 minutes. You guys are too amazing!."

    Musa, Federal University of Technology Minna
  • "I was scared at first when I saw your website but I decided to risk my last 3k and surprisingly I got my complete project in my email box instantly. This is so nice!!!."

    Ali Obafemi, Ibrahim Badamasi Babangida University, Niger State.
  • To contribute to our success story, send us a feedback or please kindly call 2348037664978.
    Then your comment and contact will be published here also with your consent.

    Thank you for choosing researchwap.com.