Project Abstract

<p> The study was carried out to determine pharmacognostic standards, phytochemical constituents,<br>evaluate the antioxidant and hepatoprotective activities of the extract and fractions of Annona<br>senegalensis Pers. (Annonaceae) by employing both the in vitro and in vivo experimental<br>models. The acute toxicity tests result showed the drug is safe at 5000mg/kg doses.<br>The effect of DPPH free radical scavenging, ABTS radical scavenging, Hydroxyl radical<br>scavenging, Hydrogen peroxide scavenging, lipid peroxidation assay, assay of catalase,<br>superoxide Dismutase assay, total protein, β-carotene bleaching, FRAP scavenging, liver and<br>superoxide anion radical scavenging activities were evaluated. Hepatoprotective effects of the<br>extract was evaluated against CCl4 induce liver damage. Carbon tetrachloride (CCl4) induce<br>hepatotoxicity was evaluated by significant increase ( p &lt; 0.05 ) in serum AST, ALT, ALP<br>activity and bilirubin level accompanied by significant decrease ( p &gt; 0.05 ) lipid peroxidation,<br>and catalase activity in liver tissue. All these parameters were also evaluated using the n–hexane,<br>ethyl acetate, methanol fractions.<br>The results showed that the extract/fractions of stem bark of A. senegalensis had better<br>antioxidant activities at high concentrations when compared to the standard. Co-administration<br>of the extract/fractions (400mg/Kg) protects the CCl4 – induced lipid peroxidation, restored<br>altered serum elevated enzymes. It showed that it is dose dependent.<br>The results obtained in the present study indicate that the stem bark is a potential source of<br>natural antioxidants. <br></p>

Project Overview

<p> 1.0 INTRODUCTION<br>There is amazing abundance of plant life in rain forest and nature has blessed the mankind with a<br>treasure of herbal remedy secrets that offer new approaches to health and healing. It is quite<br>interesting to discover that different herbs can be indicated for a vast number of health problems.<br>Therefore, a lot of scientific screening and research have been going on into investigating the<br>various constituents of plants that are responsible for a particular activity or more, despite all<br>odds. Many drugs have been discovered by the exploitation of traditional medicine since the<br>early dates of human existence (Nwaogu, 1997). Plants have invariably been a rich source for<br>new drugs and some antioxidant drugs in use today were either obtained from plants or<br>developed using their chemical structures as templates(Nwaogu, 1997).<br>Currently, there is an increasing awareness of the value of traditional medicine and the necessity<br>for improving its standard. Indeed, the Organization of African Unity (O.AU) has in the last few<br>years, held lots of international symposia and these were on a particular aspect of a subject,<br>notably medicinal plants. It has been observed that many plants contain a variety of<br>phytochemical substances, which have appreciable physiological and pharmacological actions on<br>man and animals. Researches on natural products over the years have revealed enormous<br>potentials of plants as source of medicinal agents. Plants are no longer being cultivated for food<br>alone, but also as sources of drugs.<br>Herbal medicine, which is the oldest form of healthcare known to mankind, involves the use of<br>herbs (medicinal plants) for therapeutic or medicinal purposes. Herbal medicine can be broadly<br>classified into various systems: traditional Chinese herbalism, which is part of the traditional<br>2<br>oriental medicine; Ayurveda herbalism, which is derived from Ayurveda; and Western<br>herbalism, which originally came from Greece and Rome to Europe and then spread to North and<br>South America.<br>The medicinal plants which may be leaves, stems, roots, flowers, seeds, fruits or whole plant or<br>any combination of these parts are prepared in various forms for therapeutic purposes. From a<br>scientific approach, most of the preparations are considered unscientific since they are not<br>pharmacologically authenticated or standardized and are seen as unrefined.<br>Many plants have varied pharmacological effects which have been confirmed. Extracts of<br>Digitalis spp, Colchicumautomnale, Catharanthusroseus and Peyotecactus had cardio – active,<br>anti – inflammatory, anti – neoplastic and central nervous system actions respectively. It is<br>already estimated that 122 drugs from 94 plants species have been discovered throughethno<br>botanical leads. Plants commonly used in traditional medicines assumed to be safe due to their<br>long usage in the treatment of disease according to knowledge accumulated over centuries.<br>However, recent scientific findings had shown that many plants used as food or in traditional<br>medicine are potentially toxic, mutagenic and carcinogenic (Schimmer et al., 1994).<br>Cancer chemoprevention by using antioxidant approaches has been suggested to offer a good<br>potential in providing important fundamental benefits to public health, and is now considered by<br>many clinicians and researchers as a key strategy for inhibiting, delaying, or even reversal of the<br>process of carcinogenesis. The cancer chemopreventive activities of naturally occurring<br>phytocompounds are of great interest.<br>Liver diseases such as jaundice, cirrhosis and fatty liver diseases are very common and large<br>public health problem in the world. Jaundice and hepatitis are two major hepatic disorders that<br>3<br>account for a high death rate. There is no rational therapy available for treating liver disorders<br>and management of liver diseases is still a challenge to the modern medicine. The modern<br>medicines have little to offer for alleviation of hepatic ailments whereas most important<br>representatives are of phytoconstituents .The traditional system of medicine like Ayurveda and<br>Siddha system of medicine, Unani system, Chinese system of medicine, Kampoo (Japanese)<br>system of medicine have a major role in the treatment of liver ailments.<br>Some medicinal plants are used in treatment of hepatobiliary pathologies. Many Nigerian ethno<br>botanic traditions propose a rich repertory of medicinal plants used by the population for<br>treatment of liver diseases. However, there were not enough scientific investigations on the<br>hepatoprotective activities conferred to these plants. One of such plant from Nigerian flora is<br>Annona senegalensisPers.It is believed in folkore that the fruit obtained from this multipurpose<br>plant is widely used locally in the treatment of two commonly energy deficiency syndrome<br>known as kwashiorkor and marasmus. Dalziel, (1995) made report about the plant to be of great<br>medicinal value and its used in native medicine to treat headache and body ache, eyelid swelling.<br>The stem bark of A. senegalensis is used by local populations all over Africa in treating guinea<br>worms, diarrhea and especially in northern Nigeria, gastroenteritis, snake bites, toothache,<br>respiratory infections and malaria. Awa and colleagues (2012) reported the use of leaves in the<br>treatment of pneumonia, and as a stimulant to improve health. A decoction from the roots is used<br>to stop chest colds, venereal diseases, stomach ache and dizziness (Jiofack. et al., 2010).<br>Many indigenous herbal plants of regional interest have been used popularly as folk medicines<br>in Nigeria or other African countries; however, their bioactivities or pharmacological effects are<br>to be investigated.<br>4<br>1.1 PLANT PHARMACOGNOSTIC PROFILE<br>1.1.1 TAXONOMY<br>Kingdom: Plantae<br>Division: Magnoliophyta<br>Class: Magnoliopsida<br>Order: Magnoliales<br>Family: Annonaceae<br>Genus: Annona<br>Species: senegalensis<br>Authority: Pers<br>5<br>Fig 1: A. senegalensis<br>6<br>1.2.0 BOTANICAL DESCRIPTION<br>Annona senegalensis is a shrub or small tree 2 – 6m tall but may reach 11m under favorable<br>conditions, It has a bark smooth to roughish, silvery-grey or grey-brown, with leaf scars and<br>roughly circular flakes exposing paler patches of under bark. Young branches with dense, brown,<br>yellow or grey hairs that are lost later. The leaves are alternate, simple, oblong, ovate or elliptic,<br>6 – 18.5cm x 2.5 – 11.5cm, green to bluish green, almost without hairs on top, but after with<br>brownish hairs or underside. They have net veination which may be green or reddish on both<br>surfaces. The apex is round or slightly notched with base square to slightly lobed base. The<br>margined is entire; petiole short, 0.5 – 2.5cm thick set (Ketende et al. 1995). Flowers up to 5cm<br>in diameter, on stalk, 2cm long, solitary or in groups of 2 – 4, arising above the leaf axils; 6<br>fleshly cream to yellow petals in 2 whorls, greenish outside, creamy or crimson, 0.8 – 1.5cm x<br>0.9 – 1.1cm, glabrous or minutely papillose within; 3 in number, free, smaller than the petals, 3-<br>4×4 – 5cm; stamens 1.7 – 2.5mm long. Fruits formed from many fused compels, fleshy, lumpy,<br>egg shaped, 2.5 – 5×2.5 – 4cm, ovoid or globose; unripe fruit green turning yellow to orange or<br>ripening stalk 1.5 – 5cm long; seeds numerous, cylindrical, oblong, orange brown. The genus<br>name, “Annona”, is from the Latin word “anon”, meaning “yearly produce”, referring to the<br>production habits of fruits of the various species in the genus. The specific name means “of<br>Senegal”, which is where the type specimen was collected (Beentje, 1994).<br>7<br>1.2.1 REPORTED ACTIVE CONSTITUENTS<br>A. senegalensis has been shown to contain a lot of constituents which are responsible for its<br>various pharmacological properties. These secondary metabolites which include; alkaloids (-);<br>roemerine, an aporphine), tannins, flavonoids, resins, glycosides, carbohydrates and<br>saponins.Others constituents reported include aliphatic ketones, alkanes, fatty acids, and sterols<br>from the leaves, monoterpenoids and sesquiterpenoids from the essential oil of the leaves and<br>fruits, amino acids from the stem bark; and ent-kaurenoids from the root back (Silva,et al, 1995).<br>1.2.2 ETHNOMEDICINAL USES OF A. SENEGALENSIS<br>Several plant parts of A. senegalensis are used in traditional medicine in various countries of<br>tropical Africa for the treatment of many diseases and symptoms including: cancer, convulsions,<br>diarrhea, dysentery, Malaria fever and filariasis, male impotency, pain of the chest and intestines,<br>inflammations, trypanosomiasis, venereal diseases and snake bite. Root extracts of A.<br>senegalensis have been found to exhibit antineoplastic activity in mice bearing sarcoma 180<br>ascites tumor cell, and antiprotozoal activity in mice infected with Trypanosomabrucei<br>(Silva,1995).<br>. The leaves are sometimes used as vegetables, while the edible white pulp of the ripe fruit has a<br>pleasant, pineapple like taste (FAO, 1983). An effective insecticide is obtained from the bark.<br>The bark is used for treating guinea worms and other worms, gastroenteritis, toothache and<br>respiratory infections. Gum from the bark is used in sealing cuts and wounds. The leaves are<br>used for treating pneumonia and as a tonic to promote general wellbeing. Roots are used for<br>stomach-ache, chest colds and dizziness. Various plant parts are combined for treating<br>dermatological diseases and ophthalmic disorders. In South Africa, roots are said to cure<br>8<br>madness, and in Mozambique, they are fed to small children to induce them to forget the breast<br>and thus hasten weaning. It has also been claimed that leaves picked on a Thursday morning and<br>thrown over the right shoulder brings good luck (Anon 1986).<br>1.3.0 GENERAL REVIEW OF ANTIOXIDANT AND HEPATOPROTECTIVE STUDIES<br>1.3.1 OXIDATIVE STRESS (OS)<br>Oxidative stress (OS) is a general term used to describe the steady state level of oxidative<br>damage in a cell, tissue, or organ, caused by the reactive oxygen species (ROS). This damage can<br>affect a specific organ or the entire organism. ROS such as free radicals and peroxides, represent<br>a class of molecules that are derived from the metabolism of oxygen and exist inherently in all<br>aerobic organisms.<br>OS is caused by an imbalance between the production of reactive oxygen species and detoxifier<br>(antioxidants). All forms of life in normal state maintain an equilibrium redox reaction.<br>Distortion of this normal redox state can cause toxic effects through the production of peroxides<br>and free radicals that can damage components of the cell, including proteins, lipids, and DNA<br>(Aroma 1993).<br>The level of oxidative stress is determined by the imbalance between the rate at which oxidative<br>damage is induced and the rate at which it is efficiently repaired and removed. The rate at which<br>damage is caused is determined by how fast the reactive oxygen species are generated and then<br>inactivated by endogenous defense agents called antioxidants. The rate at which damage is<br>removed is dependent on the level of repair enzymes. The determinants of oxidative stress are<br>regulated by an individual’s unique heredity factors, as well as his/her environment and<br>characteristic lifestyle. Unfortunately, under the present day life-style conditions many people<br>9<br>run an abnormally high level of oxidative stress that could increase their probability of early<br>incidence of decline in optimum body functions (Aroma 1993).<br>In humans, oxidative stress is involved in many diseases, such as atherosclerosis, Parkinson’s<br>disease and Alzheimer’s disease and it may also be important in ageing. However, reactive<br>oxygen species can be beneficial, as they are used by the immune system as a way to attack and<br>kill pathogens and as a form of cell signaling (Rice-Evans, et al., 1995).<br>In chemical terms, oxidative stress is a large increase in the cellular reduction potential, or a<br>large decrease in the reducing capacity of the cellular redox couples, such as glutathione. The<br>effects of oxidative stress depend upon the size of these changes, with a cell being able to<br>overcome small perturbations and regain its original state (Seis, 1997).<br>A particularly destructive aspect of oxidative stress is the production of reactive oxygen species,<br>which include free radicals and peroxides. Some of the less reactive of these species (such as<br>superoxide) can be converted by redox reactions with transition metals or other redox cycling<br>compounds including Quinone into more aggressive radical species that can cause extensive<br>cellular damage (Valko, et al., 2005). Most of these oxygen- derived species are produced at a<br>low level by normal aerobic metabolism and the damage they cause to cells is constantly<br>repaired. However, under the severe levels of oxidative stress that cause necrosis, the damage<br>causes ATP depletion, preventing controlled apoptotic death and causing the cell to simply fall<br>apart (Lelli, et al., 1998).<br>10<br>1.3.2 REACTIVE OXYGEN SPECIES (ROS)<br>Reactive oxygen species are chemical species which are responsible for toxic effects in the body<br>through various tissue damages. They are formed either by the loss of a single electron from a<br>non-radical or by the gain of a single electron by a non-radical.<br>Examples of ROS are listed in table 1<br>Table 1: Description of oxidants<br>Oxidant Description<br>.O2 superoxide anion One-electron reduction state of O2 , formed in many<br>autoxidation reactions and by the electron transport chain.<br>Rather unreactive but can release Fe2+ from iron-sulphur<br>proteins and ferritin. Undergoes dismutation to form H2O2<br>spontaneously or by enzymatic catalysis and is a precursor<br>for metal-catalyzed .OH formation.<br>H2O2, hydrogen peroxide Two-electron reduction state, formed by dismutation of .O2<br>–<br>or by direct reduction of O2. Lipid soluble and thus able to<br>diffuse across membranes.<br>.OH, Hydroxyl radical Three-electron reduction state formed by Fenton reaction<br>and decomposition of peroxynitrite. Extremely reactive, will<br>attack most cellular components.<br>ROOH, organic hydro peroxide Formed by radical reactions with cellular components such as<br>lipids and nucleobases.<br>RO., alkoxy and ROO., Peroxy<br>radicals.<br>Oxygen centered organic radicals. Lipid forms precipitates in<br>lipid peroxidation reactions. Produced in the presence of<br>oxygen by radical addition to double bonds or hydrogen<br>abstraction.<br>HOCL, hypochlorous acid. Formed from H2O2 by myeloperoxidase. Lipid soluble and<br>highly reactive. Will readily oxidize protein constituents,<br>including thiol groups, amino groups and methionine.<br>OONO-, peroxynitrite Formed in a rapid reaction between .O2<br>– and NO. Lipid soluble<br>and similar in reactivity to hypochlorous acid. Protonation<br>forms peroxynitrous acid, which can undergo hemolytic<br>cleavage to form hydroxyl radical and nitrogen dioxide.<br>The most important source of reactive oxygen species under normal conditions in aerobic<br>organisms is probably the leakage of activated oxygen from mitochondria during normal<br>oxidative respiration. Other enzymes capable of producing superoxide are xanthine oxidase,<br>reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and cytochromes<br>11<br>P450. Hydrogen peroxide is produced by a wide variety of enzymes including monoxygenases<br>and oxidases. Reactive oxygen species play important roles in cell signaling, a process termed<br>redox signaling. Thus, to maintain proper cellular homeostasis, a balance must be struck between<br>reactive oxygen production and consumption (Aroma 1993).<br>Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive<br>anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce<br>free radicals or are chemically activated by them. Under normal conditions, ROS are cleared<br>from the cell by the action of superoxide dismutase (SOD), catalase, or glutathione (GSH)<br>peroxide. The main damage to cells results from the ROS-induced alteration of macromolecules<br>such as polyunsaturated fatty acids in membrane lipids, essential proteins, and DNA (Nishikimi,<br>et al 1972).<br>Exogenous sources of ROS include exposure to cigarette smoke, environmental pollutants such<br>as emission from automobiles and industries, consumption of alcohol in excess, asbestos,<br>exposure to ionizing radiation, and bacteria, fungi or viral infections.<br>Poor nutrition in general contributes to OS. When the body is fed poorly, it slowly starves and all<br>of its systems suffer. Weak organ systems are prime targets for free radical attack.<br>Even psychological and emotional stress can contribute to OS. When the body is under stress, it<br>produces certain hormones that generate free radicals. Moreover, the liver must eventually<br>detoxify them and that process also generates free radicals.<br>Heightened OS has also been observed in athletes after intensive workouts due to the physical<br>stress placed on the body. Both physical and emotional stress also prompts the release of<br>12<br>endogenous cortisol, an adrenal hormone that reduces inflammation, but also suppresses the<br>immune system (Seis, 1997).<br>Endurance exercise can increase oxygen utilization from 10 to 20 times over the resting state.<br>This greatly increases the generation of free radicals, prompting concern about enhanced damage<br>to muscles and other tissues (Rice – Evans et al 1995).<br>Metals such as iron, copper, chromium, vanadium and cobalt are capable of redox cycling in<br>which a single electron may be accepted or donated by metal ion or metal. The most important<br>reactions are probably Fenton’s reaction and the Haber-Weiss reaction, in which hydroxyl<br>radical is produced from reduced iron and hydrogen peroxide (<a target="_blank" rel="nofollow" href="http://www.en.wikipedia.org/oxidativestress)">www.en.wikipedia.org/oxidativestress)</a>.<br>The hydroxyl radical then can lead to modifications of amino acids (e.g. meta-tyrosine<br>and ortho-tyrosine formation from phenylalanine, carbohydrates, initiate lipid peroxidation, and<br>oxidize nucleobases. Most enzymes that produce reactive oxygen species contain one of these<br>metals. The presence of such metals in biological systems in an uncomplexed form can<br>significantly increase the level of oxidative stress (Valko, et al2005).<br>Certain organic compounds in addition to metal redox catalyst can also produce reactive oxygen<br>species. One of the most important classes of these is the quinones. Quinones can redox cycle<br>with their conjugate semiquinones and hydroquinones, in some cases catalyzing the production<br>of superoxide from dioxide or hydrogen peroxide from superoxide (Valko et al2005).<br>1.3.3 LIPID PEROXIDATION AND FREE RADICALS<br>Lipid peroxidation refers to the oxidation degradation of lipids. It is the process whereby free<br>radicals “steal” electrons from the lipids in cell membranes, resulting in cell damage. Lipid<br>13<br>hydroperoxides are non-radical intermediates derived from unsaturated fatty acids,<br>phospholipids, glycolipids, cholesterol esters and cholesterol itself. Their formation occurs in<br>enzymatic or non –enzymatic reactions involving activated chemical species known as “reactive<br>oxygen species” (ROS) which are responsible for toxic effects in the body via various tissue<br>damages. They are formed either by the loss of a single electron from a non-radical or by the<br>gain of a single electron by a non-radical. They can easily be formed when a covalent bond is<br>broken if one electron from each of the pair shared remains with each atom, this mechanism is<br>known as hemolytic fission. In water, this process generates the most reactive species, hydroxyl<br>radicals OH. Chemists know well that combustion which is able at high temperature to rupture<br>C – C, C – H or C – O bonds is a free radical process. The opposite of this mechanism is the<br>heterolytic fission in which, after a covalent break, one atom receives both electrons (this gives a<br>negative charge) while the other remains with a positive charge. This process proceeds by a free<br>radical chain reaction mechanism. It most often affects polyunsaturated fatty acids, because they<br>contain multiple double bonds in between which lies methylene –CH2- groups that possess<br>especially reactive hydrogen (McCay et al., 1984).<br>As with any radical reaction, the reaction consists of three major steps: initiation, propagation<br>and termination. Initiation is the step whereby a radical is produced. The initiators in living cells<br>are most notably reactive oxygen species (or ROS), such as OH, which combines with a<br>hydrogen atom to make water and a fatty acid radical(<a target="_blank" rel="nofollow" href="http://www.wikipedia.org/wiki/lipidperoxidation)">www.wikipedia.org/wiki/lipidperoxidation)</a>.<br>The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen,<br>thereby creating a peroxyl-fatty acid radical. This too is an unstable species that’s reacts with<br>another free fatty acid producing a different fatty acid radical and a hydrogen peroxide or cyclic<br>14<br>peroxide if it had reacted with itself. This cycle continues as the new fatty acid radical reacts in<br>the same way. This is the propagation stage(<a target="_blank" rel="nofollow" href="http://www.wikipedia.org/wiki/lipid-peroxidation)">www.wikipedia.org/wiki/lipid-peroxidation)</a>.<br>In termination stage a radical reacts with another radical, which is why the process is called a<br>“chain reaction mechanism”. The radical reaction stops when two radicals react and produce a<br>non-radical species. This happens only when the concentration of radical species is high enough<br>for there to be a high probability of two radicals actually colliding. Living organisms have<br>evolved different molecules to catch free radicals and protect the cell membrane. One important<br>such antioxidant is alpha-tocopherol, also known as vitamin E (<a target="_blank" rel="nofollow" href="http://www.wikipedia.org/wiki/lipidperoxidation)">www.wikipedia.org/wiki/lipidperoxidation)</a>.<br>Free radicals are highly unstable molecules that interact quickly and aggressively with other<br>molecules in our bodies to create abnormal cells. Free radicals are atoms or groups of atoms with<br>an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain<br>molecules. Their instability causes them to react almost instantly with any substance in their<br>vicinity. Oxygen, or oxyl, free radicals are especially dangerous. Once formed, these highly<br>reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the<br>damage they can do when they react with important cellular components such as DNA, or the<br>cell membrane; enzymes. Cells may function poorly or die if this occurs. They are capable of<br>penetrating into the DNA of a cell and damaging its “blueprint” so that the cell will produce<br>mutated cells that can the replicate without normal controls. They accelerate aging and contribute<br>to the development of many diseases, including cancer and heart disease (Zhang, et al, 1993).<br>Surprisingly, however, free radicals are involved in many cellular functions and are a normal part<br>of living. When, for example, mitochondrion within cell burns glucose for fuel, the mitochondria<br>15<br>oxidize the glucose and in so doing generates free radicals. White blood cells also use free<br>radicals to attack and destroy bacteria, viruses and virus-infected cells. The detoxifying actions<br>of the liver also require free radicals (Lennon, et al., 1991).<br>It is important to note that free radicals are also released in the body from the breaking down or<br>detoxification of various chemical compounds; drugs, artificial food colorings and flavorings,<br>smog, preservatives in processed foods, alcohol, cigarette smoke, chlorinated drinking water,<br>pesticides, radiation, cleaning fluids, heavy metals such as cadmium and lead, and assorted<br>chemicals such as solvent traces found in processed foods and aromatic hydrocarbons such as<br>benzene and naphthalene (found in moth balls). Additionally, certain foods contain free radicals<br>which when eaten, enter the body and damage it. The major sources of dietary free radicals are<br>chemically altered fats from commercial vegetable oils, vegetable shortening and all oils heated<br>to very high temperatures (Buege, and Aust, 1978).<br>Some free radicals arise normally during metabolism. Sometimes the body’s immune system’s<br>cells purposefully create them to neutralize viruses and bacteria. However, environmental factors<br>such as pollution, radiation, cigarette smoke and herbicides can also spawn free radicals (Nathan<br>et al., 2000).<br>Because it is not possible to directly measure free radicals in the body, scientists have<br>approached the questions of how effectively can athletes defend against the increased free<br>radicals from exercise by measuring the by-products that result from free radical reactions. If the<br>generation of free radicals exceeds the antioxidant defenses then one would expect to see more<br>of these by-products. These measurements have been performed in athletes under a variety of<br>conditions (Ellman, 1959).<br>16<br>Several interesting concepts have emerged from these types of experimental studies. Regular<br>physical exercise enhances the antioxidant defense system and protects against exercise induced<br>free radical damage. This is an important finding because it shows how smart the body is about<br>adapting to the demands of exercise. These changes occur slowly over time and appear to<br>parallel other adaptations to exercise.<br>On the other hand, intense exercise in untrained individuals overwhelms defenses resulting in<br>increased free radical damage. Thus, the “weekend warrior” who is predominantly sedentary<br>during the week but engages in vigorous bouts of exercise during the weekend may be doing<br>more harm than good. To this end there are many factors that may determine whether exercise<br>induced free radical damage occurs, including degree of conditioning of the athlete, intensity of<br>exercise and diet (Sies, 1997).<br>Normally, the body can handle free radicals, but if antioxidants are unavailable, or if the freeradical<br>production becomes excessive, damage can occur. Of particular importance is that free<br>radical damage accumulates with age.<br>1.3.4 Biological uses of Reactive Oxygen species<br>The immune system uses the lethal effects of oxidants as a central part of its mechanism of<br>killing pathogens; with activated phagocytes producing both ROS and reactive nitrogen species<br>(Nathan, et al., 2000). Although the use of these highly reactive compounds in the cytotoxic<br>response of phagocytes causes damage to host tissue, the non-specificity of these oxidants is an<br>advantage since they will damage almost every part of their target cell (Rice-Evans, et al.,<br>1995).this prevents a pathogen from escaping this part of immune response by mutation of a<br>single molecular target.<br>17<br>More recently, it has become apparent that ROS also have important roles as signaling<br>molecules. A complex network of enzymatic and small molecule antioxidants controls the<br>concentration of ROS and repairs oxidative damage, and research is revealing the complex and<br>subtle interplay between ROS and antioxidants in controlling plant growth, development and<br>response to the environment.<br>1.3.5 Consequences of Oxidative Stress.<br>Oxidative stress contributes to tissue injury following irradiation and hyperoxia. It has been<br>implicated in disease states, such as neurodegenerative diseases including Lou Gehrig’s disease<br>(aka MND or ALS), Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and aging.<br>Oxidative stress is thought to be linked to certain cardiovascular disease, since oxidation of low<br>density lipoprotein (LDL) in the vascular endothelium is a precursor to plaque formation.<br>Oxidative stress also plays a role in the ischemic Cascade due to oxygen reperfusion injury<br>following hypoxia. This cascade includes both strokes and heart attacks.<br>Other disease conditions associated with oxidative stress include: Diabetes, pancreatitis, liver<br>damage, and leaky gut syndrome, hypertension and multiple sclerosis, atherosclerosis (Steinberg,<br>et al., 1989), coronary thrombosis, asthma, emphysema, chronic pulmonary disease, cataracts,<br>retinopathy, macular degeneration, rheumatoid arthritis (Aroma, 1993), glomerulonephritis,<br>vitiligo, wrinkles (Pryor W.A., 1991), cancer, autoimmune diseases, inflammatory states<br>(Symons and Dowling, 1987), AIDS and Lupus (Montagnier, Oliveier, and Pasquier, 1998).<br>However, more severe oxidative stress can cause cell death and even moderate oxidation can<br>trigger apoptosis, while more intense stresses may cause necrosis (Lennon, et al., 1991).<br>18<br>1.3.6 ANTIOXIDANTS<br>To prevent free radical damage the body has a defense system of antioxidants. Antioxidants are<br>intimately involved in the prevention of cellular damage the common pathway for cancer, aging,<br>and a variety of diseases. Fortunately, the body maintains a sophisticated system of chemical and<br>biochemical antioxidants scavenge free radicals, that is, they stabilize the unstable free radicals<br>by giving them the electron they need to “calm down”. The antioxidants are usually consumed or<br>used up in this process, i.e., they sacrifice themselves.<br>Antioxidants are molecules that can safely interact with free radicals and terminate the chain<br>reaction before vital molecules are damaged. Although there are several enzyme systems within<br>the body that scavenge free radicals, the principle micronutrient (vitamins) antioxidants are<br>vitamin E, beta-carotene, and vitamin C. Additionally, Selenium, a trace metal that is required<br>for proper function of one of the body’s antioxidant enzyme systems, is sometimes included in<br>this category. The body cannot manufacture these micronutrients so they must be supplied in the<br>diet. Therefore the main antioxidants are vitamins A, E, and C, beta-carotene, glutathione,<br>bioflavonoids, selenium, Zinc, CoQ10 (ubiquinone), and various phyto-chemicals from herbs<br>and foods. Green tea, for example, is rich in polyphenols-powerful antioxidants that help fight<br>cancer.<br>The best studied cellular antioxidants are the enzymes superoxide dismutase (SOD), catalase,<br>and glutathione peroxidase. Less well studied (but probably just as important) enzymatic<br>antioxidants are the peroxiredoxins and the recently discovered sulfiredoxin. Other enzymes that<br>have antioxidant properties (though this is not their primary role) include paraoxonase,<br>gluthione-S transferases, and aldehyde dehydrogenases.<br>19<br>Biochemical antioxidants not only scavenge free radicals, but also inhibit their formation inside<br>the body. These include lipoic acid, and repair enzymes such as catalase, superoxide dismutase<br>(SOD), glutathione peroxidase. Melatonin, a hormone produced by the pineal gland, is also a<br>potent antioxidant. Cholesterol, produced by the liver, is another major antioxidant, which the<br>body uses to repair damaged blood vessels. It is probably for this reason that serum cholesterol<br>levels rise as people age. With age comes more free radical activity and in response the body<br>produces more cholesterol to help contain and control the damage (Seis, 1997).<br>Of all the antioxidants, glutathione appears to be pivotal. Made up of three amino acids (cysteine,<br>glycine, and glutamic acid), glutathione is part of the antioxidant enzyme glutathione peroxidase<br>and is the major liver antioxidant. It is a basic tenet of natural medicine that health cannot exist if<br>the liver is intoxicated. Not surprisingly, extremely low levels of glutathione are found in people<br>suffering from severe OS. People with AIDS, cancer and Parkinson’s disease, for example,<br>typically have low glutathione levels.<br>As noted earlier, oxidative stress occurs when the amount of free radicals in the body exceeds its<br>pool of available antioxidants. Obviously, knowing the varied sources of free radicals and<br>avoiding them in an important part of minimizing their harmful effects.<br>Diet can be a major source of free radical stressors with processed or highly heated oils being the<br>main offenders. Replace these harmful fats with natural, cold pressed oils such as olive oil<br>(which can be used for cooking) and small amounts of flax oil or walnut oil (which should never<br>be heated). Food grade, unrefined coconut oil and organic butter are also excellent choices,<br>especially for cooking. Both of these naturally saturated fats are rich in certain fatty acids that<br>have proven activity against bacteria, harmful yeasts, fungi and tumor cells.<br>20<br>Additionally, since saturated fats (from animal foods and the tropical oils) and monounsaturated<br>oils (from olive oil and cold-pressed nut oils) are more chemically stable, they are much less<br>susceptible to oxidation and rancidity than their polyunsaturated analogues, which are mostly<br>found in vegetable oils. As a general rule, then, although the body does require a small amount of<br>naturally occurring polyunsaturated oils in the diet each day, it is best not to consume too much<br>of them as they are more prone to free radical attack in the body. As Linus Pauling, noted: “A<br>diet high in unsaturated fatty acids, especially the polyunsaturated ones, can destroy the body’s<br>supply of vitamin E and cause muscular lesions, brain lesions, and degeneration of blood vessels.<br>Care must be taken not to include a large amount of polyunsaturated oil in the diet” (Linus<br>Pauling, 1998).<br>The best food sources for polyunsaturated are fish, flax oil, sesame oil, walnut oil and dark<br>green, leafy vegetables. One caveat: canola oil is not recommended due to its chemical instability<br>and its content of trans-fatty acids (TFAs), formed during processing. TFAs are increasingly<br>being linked with cancer, immune system dysfunction and heart disease.<br>21<br>A. VITAMIN C<br>fig.2<br>L – Ascorbic acid<br>Ascorbic acid is a water- soluble vitamin present in citrus fruits and juices, green peppers,<br>cabbage, spinach, broccoli, kale, cantaloupe, kiwi, and strawberries. The RDA is 60mg per day.<br>Intake above 2000 mg may be associated with adverse side effects in some individuals. Vitamin<br>C is the most abundant water-soluble antioxidant in the body and acts primarily in cellular fluid.<br>It is of particular note in combating free-radical formation caused by pollution and cigarette<br>smoke. Also helps return Vitamin E to its active form (Hickey, and Roberts, 2004).<br>The vitamins C and E are thought to protect the body against the destructive effects of free<br>radicals. Antioxidants neutralize free radicals by donating one of their own electrons, ending the<br>electron- “stealing” reaction. The antioxidant nutrients themselves don’t become free radicals by<br>donating an electron because they are stable in either form. They act as scavengers, helping to<br>prevent cell and tissue damage that could lead to cellular damage and disease (Padayatty,et al.,<br>2003).<br>22<br>B. BETA-CAROTENE<br>fig.3<br>Carotene is a terpene, synthesized biochemically from eight isoprene units. It comes in two<br>primary forms designated by characters from the Greek alphabet: alpha- carotene (-carotene) and<br>beta-carotene (-carotene). Gamma, delta and epsilon (- carotene) also exist. Beta-carotene is<br>composed of two retinyl groups, and is broken down in the mucosa of the small intestine by<br>Beta-carotene dioxygenase to retinol, a form of vitamin A. carotene can be stored in the liver and<br>converted to vitamin A as needed, thus making it a provitamin.<br>Beta-carotene is a precursor to vitamin A (retinol) and is present in liver, egg yolk, milk, butter,<br>spinach, carrots, squash, broccoli, yams, tomato, cantaloupe, peaches, and grains. Because betacarotene<br>is converted to vitamin A by the body, there is no set requirement. Instead the RDA is<br>expressed as retinol equivalents (RE), to clarify the relationship. (NOTE: Vitamin A has no<br>antioxidant properties and can be quite toxic when taken in excess). In people who smoke, betacarotene<br>may increase cardiovascular mortality (Todd, et al., 1999, Omenn, et al., 1998).in men<br>who smoke and have had a prior myocardial infarction (MI), the risk of fatal coronary heart<br>23<br>disease increases by as much as 43% with low doses of beta-carotene. There are some evidence<br>that beta-carotene in combination with selenium, vitamin C and vitamin E might lower highdensity<br>lipoprotein 2 (HDL2) cholesterol levels. HDL levels are protective so this considered<br>being a negative effect. Dizziness, reversible yellowing of palms, hands, or soles of feet and to a<br>lesser extent the face (called carotenoderma) can occur with high doses of beta-carotene. Loose<br>stools, diarrhea, unusual bleeding or bruising and joint pain have been reported.<br>C. GLUTATHIONES<br>fig. 4<br>Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-molecularweight<br>thiol within cells. Two cytosolic enzymes, gamma-glutamylcysteine synthetase and<br>glutathione synthetase catalyze the synthesis of glutathione from glutamate, cysteine, and<br>glycine. Compelling evidence shows that glutathione synthesis is regulated primarily by gammaglutamylcysteine<br>synthetase activity, cysteine availability, and glutathione feedback inhibition.<br>Animal and human studies demonstrate that adequate protein nutrition is crucial for the<br>maintenance of glutathione homeostasis.<br>24<br>In aerobic cells, free radicals are constantly produced mostly as reactive oxygen species. Once<br>produced, free radicals are removed by antioxidant defenses including the enzymes catalase,<br>glutathione peroxidase, and superoxide dismutase. Reactive oxygen species, including nitric<br>oxide and related species, commonly exert a series of useful physiological effects. Imbalance<br>between prooxidant and antioxidant defenses in favor of prooxidants results in oxidative stress,<br>this results in damage to lipids, proteins, and nucleic acids. Alone or in combination with<br>primary factors, free radicals are involved in the cause of hundreds of diseases.<br>Glutathione – or L Glutathione – is a powerful antioxidant found within every cell. Glutathione<br>plays a role in nutrient metabolism, and regulation of cellular events including gene expression,<br>DNA and protein synthesis, cell growth, and immune response. Glutathione taken as a<br>supplement may not be able to cross the cell membrane and thus may not be effective. Consider<br>acetylcysteine instead because it is the N-acetyl derivative of the amino acid, L-cysteine, and is a<br>precursor in the formation of the antioxidant glutathione in the body. The thiol (sulfhydryl)<br>group confers antioxidant effects and is able to reduce free radicals and also acetylcysteine is a<br>good alternative since it can help produce more glutathione.<br>This antioxidant, made from the combination of three amino acids cysteine, glutamate, and<br>glycine, forms part of the powerful natural antioxidant glutathione peroxidase that is found in our<br>cells. Glutathione peroxidase plays a variety of roles in cells, including DNA synthesis and<br>repair, metabolism of toxins and carcinogens, enhancement of the immune system, and<br>prevention of fat oxidation. However, glutathione is predominantly known as an antioxidant<br>protecting our cells from damage caused by the free radical hydrogen peroxide. Glutathione also<br>helps the other antioxidants in cells stay in their active form. Brain glutathione levels have been<br>found to be lower in patients with Parkinson’s disease (Zhang, 1993).<br>25<br>Glutathione is found in foods, particularly fruits, vegetables and meats. Cyanohydroxybutene, a<br>chemical found in broccoli, cauliflower, Brussels sprouts and cabbage, is also thought to increase<br>glutathione levels. Various herbs for instance cinnamon and cardamom have compounds that can<br>restore healthy levels of glutathione. Although glutathione is available in pill form over the<br>counter, its utilization by the body is questionable since we don’t know if it can easily enter cells,<br>even after it is absorbed in the bloodstream. Certain nutrients help raise tissue levels of<br>glutathione including acetylcysteine, methyl donors, alpha lipoic acid, polyphenols such as<br>pycnogenol, and vitamin B12 (Silva, et al., 1995).<br>An excellent review article in the April 1998 issue of Alternative Medicine Review summarizes<br>the known effects of acetylcysteine. The author writes, “N- acetylcysteine is an excellent source<br>of sulfhydryl groups, and is converted in the body into metabolites capable of stimulating<br>glutathione synthesis, promoting detoxification, and acting directly as a free radical scavenger.<br>Acetylcysteine has historically been as a mucolytic [mucus dissolving] agent in a variety of<br>respiratory illness; however, it appears to also have beneficial effects in conditions characterized<br>by decreased glutathione or oxidative stress, such as HIV infection, cancer, heart disease, and<br>cigarette smoking”. The frequent use of acetaminophen (paracetamol) depletes glutathione<br>peroxidase levels. There appear to be feedback inhibition in glutathione synthesis. This means<br>that if glutathione levels are excessively increased with the help of the nutrients, the body may<br>decrease its natural production (Kelly, 1998).<br>Glutathione is solid in pills with dosages ranging from 50 to 250mg. Glutathione is a promising<br>antioxidant. However, due to the inconsistence in the medical literature on the ability of<br>glutathione to enter tissues and cells when ingested orally, its beneficial effect to oral dosing may<br>be questionable. Oral administration is poorly tolerated, owing to high doses required (due to low<br>26<br>oral bioavailability), very unpleasant taste and odor, and adverse effects (particularly nausea and<br>vomiting). In a research conducted by Baker, it was concluded that oral N-acetylcysteine was<br>identical in bioavailability to cysteine precursors. Glutathione deficiency contributes to oxidative<br>stress, which plays a key role in aging and the worsening of many diseases including<br>Alzheimer’s disease, Parkinson’s disease, liver disease, cystic fibrosis, sickle cell anemia, HIV,<br>AIDS, cancer, heart attack, and diabetes. The concentration of glutathione declines with age and<br>in some age-related diseases (Liu, et al., 2004).<br>Staying on top of oxidative stress is a necessity in our increasingly toxic world. Taking care to<br>avoid those toxins as much as possible and to enrich our diets with life-giving antioxidants is a<br>wise step to take in our endless quest for wellness.<br>FOOD SOURSES OF ANTIOXIDANTS<br>CoQ10 (ubiquinone): Beef heart, beef liver, sardines, spinach, peanuts.<br>Beta carotene: All orange and yellow fruits and vegetables; dark green vegetables.<br>Zinc: Oysters, herring, lamb, whole grains.<br>Selenium: Butter, meats, seafood, whole grain.<br>Vitamin A: Cold liver oil, butter, liver, all oily fish.<br>Vitamin E: Cold-pressed, unrefined nut and seed oils; wheat germ oil.<br>Vitamin C: Berries, greens, broccoli, kale, kiwi, parsley, guava.<br>27<br>Glutathione (GSH): Fresh fruits and vegetables, fresh meats, low-heat dried whey.<br>Bioflavonoids: Most fruits and vegetables, buckwheat.<br>Polyphenols: Greentea,berries.<br>Herbal sources: Milk thistle, Ginkgo biloba, turmeric, curry (Padma 28, a packaged Ayurvedic<br>herbal formula, is a special blend of herbal antioxidants).<br>1.3.7 BIOTRANSFORMATION OF CARBON TETRACHLORIDE<br>Metabolism of carbon tetrachloride is initiated by cytochrome P-450 mediated transfer of an<br>electron to the C – Cl bond forming an anion radical that eliminates chloride, trichloromethyl<br>radical (Pohl et al., 1981). This radical may undergo both oxidative and reductive<br>biotransformation. The isoenzymes implicated in this process are the cytochrome P2E1,<br>cytochrome P2B1 and cytochrome P2B2 ( Gruebeleet al., 1996). Some isoforms may<br>preferentially be susceptible to degradation of carbon tetrachloride. Evidence that carbon<br>tetrachloride inactivates CYP2E, and reduces total CYP2E protein has been obtained by Dai and<br>Cederbaum (1995). When protein synthesis is blocked, inactivation and degradation of CYP2E1<br>by carbon tetrachloride are more pronounced.<br>The formation of carbon tetrachloride – cytochrome P-450 complexes has been demonstrated.<br>The most important pathway in the elimination of trichloromethyl radicals is the reaction with<br>molecular oxygen, resulting in the formation of trichloromethyl peroxyl radicals as proposed by<br>McCayet al., (1984).<br>28<br>Carbon tetrachloride has been reported to be metabolized to CO2 in the liver homogenates. The<br>biotransformation of carbon tetrachloride to carbon IV oxide in vivo has been reported by<br>Reynolds et al., (1984).<br>1.3.8 SERUM ENZYME DETERMINATIONS AS A DIAGNOSTIC TOOL<br>Normally most enzymes reside within cells, where they function in various phases of<br>intermediary metabolism and only small quantities are present in the serum. During certain acute<br>physiologic insults such as myocardial infarction or acute hepatitis, cellular content escapes with<br>extra cellular fluid and eventually reaches the serum in high concentration.<br>1.3.9 ENZYMES IN THE DIAGNOSTIC PATHOLOGY: ALT, AST AND ALP<br>I. Alanine Transaminase (ALT)<br>Alanine Transaminase (ALT) formerly called Glutamate-Pyruvate Transaminase (GPT) is an<br>enzyme present in hepatocytes (liver cells), and in less amount in kidney, heart and skeletal<br>muscle. When a cell is damaged, it leaks this enzyme into the blood, where it is measured. ALT<br>rises dramatically in acute liver damage, such as viral hepatitis than AST (Song, et al., 2010).<br>II. Aspartate Transaminase (AST)<br>Aspartate Transaminase (AST) formerly called Glutamate-Oxaloacetate Transaminase (GOT) is<br>similar to ALT in that it is another enzyme associated with liver parenchymal cells. It is raised in<br>acute liver damage, myocardial infarction, myopathies muscular disease (muscular dystrophy,<br>rhabdomyolisis) or trauma but is also present in red cells, brain, cardiac and skeletal muscles. It<br>is therefore less specific to liver disease.<br>29<br>III. Alkaline phosphatase (ALP)<br>Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts of the liver. ALP<br>levels in plasma will rise with bile duct obstruction, intra-hepatic cholestasis or infiltration<br>disease of the liver. ALP is also present in bone and placental tissue, so it is higher in growing<br>children.<br>IV. Bilirubin<br>Increased total bilirubin causes jaundice and its increased production causes hemolytic anemia<br>and internal hemorrhage. Deficiencies in bilirubin metabolism can cause cirrhosis and viral<br>hepatitis, while its deficiencies in excretion can bring about obstruction of the bile duct (Schmidt<br>and Schmidt, 1963).<br>1.4.0 Rationale of study<br>Most of the health benefits observed in people that use the extracts of AnnonasenegalensisPers.<br>(Annonaceae) stem bark for the management of many ailments are attributed to its<br>pharmacological and medicinal properties. This study is aimed at understanding the baseline<br>pharmacological and toxicological effects of the extracts of A. Senegalensis stem bark in<br>hepatotoxic and normal rats. The rationale of this work is linked to the hepatoprotective effect of<br>A.senegalensisstem bark extract on the liver (Dalziel, 1995).<br>30<br>1.4.1 Aim of study<br>u To determine pharmacognostic standards of Annona Senegalensis Pers.<br>u To determine phytochemical constituents of pulverized bark of Annonasenegalensis.<br>u To evaluate the antioxidant activities of the extract and fractions of Annona senegalensis.<br>u To evaluate the hepatoprotective activities of the extract and fractions of Annona<br>senegalensis. <br></p>