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TOTAL ANTIOXIDANT CAPACITY OF ETHANOLIC EXTRACT OF HIPPOCRETEA WELWITSCHII OLIV

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CHAPTER ONE

1.0 INTRODUCTION

Free radicals (reactive oxygen species and reactive nitrogen species) have been implicated in a large number of pathological and disease conditions as well as in the aging process (Valdez et al., 2000). Evidences have suggested that the role of these free radical species especially the reactive oxygen species in the etiology and development of these pathological conditions may be through their oxidative damage to cells. However, other researches supported the idea that the destabilization of free radical generating pathways could play a role in causes and consequence of some diseased conditions (Mates et al., 1999). The oxidative stress experienced by a tissue or cell results from the negative imbalance between the production and removal of potentially damaging reactive oxygen species (Ros). The removal rate is mostly controlled by a variety of antioxidants e.g glutathione, catalase, superoxide dismutase, Tocopherols (vitamin E, ascorbic acid (vitamin C) etc. The reduce rate of removal of reactive oxygen species may be due to reduce level of quantity and activity of these antioxidants. However, the low molecular weight antioxidants have been shown to be present in the various parts of different plants such as (khan et al., 2010). Hence plants are good sources of antioxidants to supplement the natural antioxidants of the body. Several studies have reported different total antioxidant capacity for various plants (bhalodi et al., 2008) 

1.1 AIM

The purpose of this study is to;

Determine the total antioxidant capacity of hippocratea welwitshii oliv in albino wistar rats.

1.2 LITERATURE REVIEW

1.2.1 FREE RADICALS

Free radicals are defined as molecules having an unpaired electron in the outer orbit. (Gilbert, 2000). They are generally unstable and very reactive. Examples of oxygen free radicals are superoxide (02), hydroxyl (OH), peroxyl (RO2•),alkoxyl (RO•), and hydroperoxyl (HO2• ) radicals. Nitric oxide (NO) and nitrogen dioxide (•NO2) are two nitrogen free radicals. Oxygen and nitrogen free radicals can be converted to other non-radical reactive species, such as hydrogen peroxide(H2O2), hypochlorous acid (HOCl), hypobromous acid (HOBr), and peroxynitrite (ONOO) .(Pham et al., 2008). ROS, reactive nitrogen species (RNS), and reactive chlorine species are produced in animals and humans under physiologic and pathologic conditions. (Evans et al., 2001). Thus, ROS and RNS include radical and non-radical species.



1.2.2 OXIDATIVE STRESS

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage (Halliwell and Glutteridge,2007). Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and(deoxyribonucleic acid) DNA. some reactive oxidative species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signaling. (Halliwell and Glutteridge,2007).

In humans, oxidative stress is thought to be involved in the development of cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, Sickle Cell Disease, infection (Valko et al., 2007).However, reactive oxygen species can be beneficial, as they are used by the immune system as a way to attack and kill pathogens.(Segal,2005). 

1.2.3 ANTIOXIDANTS

Antioxidants are molecules that can neutralize free radicals by accepting or donating electron(s) to eliminate the unpaired electrons of the free radical. The antioxidant molecules may directly react with the reactive radicals and destroy them, while they may become new free radicals which are less active, longer-lived and less dangerous than those radicals they have neutralized (Jian-Ming Lü et al, 2010). Cells, tissues, and body fluids are equipped with these powerful defense systems that help counteract oxidative challenge. To maintain a steady-state of metabolites and functional integrity in the aerobic environment antioxidant defense is organized at 3 principal levels of protection prevention, interception, and repair (Seis, 2007). Matching the diversity of prooxidants, the antioxidant molecules comprises a widespread array of systems which include the enzymatic (e.g superoxide dismutases, glutathione peroxidases, catalases etc) and non enzymatic (low molecular weight e.g vitamin C & E, gluthatione, etc) antioxidants 

1.2.4 FUNCTIONS/USES OF ANTIOXIDANTS 

An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols. (Sies et al., 1997).

1.2.5 ANTIOXIDANT METABOLITES 

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

The relative importance and interactions between these different antioxidants is a very complex question, with the various metabolites and enzyme systems having synergistic and interdependent effects on one another. (Chaudière et al., 1997). The action of one antioxidant may therefore depend on the proper function of other members of the antioxidant system.( Vertuani et al., 2004). The amount of protection provided by any one antioxidant will also depend on its concentration, its reactivity towards the particular reactive oxygen species being considered, and the status of the antioxidants with which it interacts.( Vertuani et al., 2004).

Some compounds contribute to antioxidant defense by chelating transition metals and preventing them from catalyzing the production of free radicals in the cell. Particularly important is the ability to sequester iron, which is the function of iron-binding proteins such as transferrin and ferritin.(Imlay et al., 2003) Selenium and zinc are commonly referred to as antioxidant nutrients, but these chemical elements have no antioxidant action themselves and are instead required for the activity of some antioxidant enzymes, as is discussed below.

Antioxidant metabolite Solubility Concentration in human serum (μM) Concentration in liver tissue (μmol/kg)

Ascorbic acid (vitamin C) Water 50 – 60 260 (human) 

GlutathioneWater 4

6,400 (human) 

Lipoic acidWater 0.1 – 0.7 4 – 5 (rat) 

Uric acidWater 200 – 400 1,600 (human) 

CarotenesLipid β-carotene: 0.5 – 1 retinol (vitamin A): 1 – 3 5 (human, total carotenoids) 

α-Tocopherol (vitamin E) Lipid 10 – 40 50 (human) 

Ubiquinol (coenzyme Q) Lipid 5 200 (human) 

1.2.6 URIC ACID

Uric acid is the highest concentration antioxidant in human blood. Uric acid (UA) is an antioxidant oxypurine produced from xanthine by the enzyme xanthine oxidase, and is an intermediate product of purine metabolism. (Enomoto et al., 2005). In almost all land animals, urate oxidase further catalyzes the oxidation of uric acid to allantoin,(Wu x et al., 1989), but in humans and most higher primates, the urate oxidase gene is non-functional, so that UA is not further broken down.(Wu x et al., 1989) Studies of high altitude acclimatization support the hypothesis that urate acts as an antioxidant by mitigating the oxidative stress caused by high-altitude hypoxia.(Baillie et al., 2007).The administration of UA is therapeutic in experimental allergic encephalomyelitis (EAE), an animal model of MS.(Hooper et al., 2000).

Uric acid has the highest concentration of any blood antioxidant (Glantzounies et al., 2005), and provides over half of the total antioxidant capacity of human serum. (Becker et al., 1993). Uric acid's antioxidant activities are also complex, given that it does not react with some oxidants, such as superoxide, but does act against peroxynitrite,(Sautin et al., 2008) peroxides, and hypochlorous acid. Concerns over elevated UA's contribution to gout must be considered as one of many risk factors. (Eggebeen et al., 2007). By itself, UA-related risk of gout at high levels (415–530 μmol/L) is only 0.5% per year with an increase to 4.5% per year at UA supersaturation levels (535+μmol/L).(Campion et al., 1987).Many of these aforementioned studies determined UA's antioxidant actions within normal physiological levels, and ,(Sautin et al., 2008) some found antioxidant activity at levels as high as 285 μmol/L.(Nazarewicz et al., 2007).

1.2.7 ASCORBIC ACID (vitamin C)

Ascorbic acid or "vitamin C" is a monosaccharide oxidation-reduction (redox) catalyst found in both animals and plants. As one of the enzymes needed to make ascorbic acid has been lost by mutation during primate evolution, humans must obtain it from the diet; it is therefore a vitamin.(Simimoff et al., 2001). Most other animals are able to produce this compound in their bodies and do not require it in their diets.(Linster et al., 2007). Ascorbic acid is required for the conversion of the procollagen to collagen by oxidizing proline residues to hydroxyproline. In other cells, it is maintained in its reduced form by reaction with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxins.(Meister et al., 1994). Ascorbic acid is a redox catalyst which can reduce, and thereby neutralize, reactive oxygen species such as hydrogen peroxide. (Padayatty et al., 2003). In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the redox enzyme ascorbate peroxidases, a function that is particularly important in stress resistance in plants. (Shigeoka et al., 2002). Ascorbic acid is present at high levels in all parts of plants and can reach concentrations of 20 millimolar in chloroplasts. (Simimoff et al., 2000).

1.2.8 POLYPHENOLS

Polyphenols antioxidants (resveratrol, flavonoids) are characterized by the presence of several phenol functional groups. Phenolic compounds such as flavonoids are ubiquitous within the plant kingdom: approximately 3,000 flavonoids substances have been described.(Briviba et al., 1994). In plants, flavonoids serve as protectors against a wide variety of environmental stresses while, in humans, flavonoids appear to function as biological response modifiers .Flavonoids have been demonstrated to have anti-inflammatory, antiallergenic ,anti-viral, anti-aging, and anti-carcinogenic activity.(Cody et al., 1986).The broad therapeutic effects of flavonoids can be largely attributed

to their antioxidant properties. In addition to an antioxidant effect,

flavonoids compounds may exert protection against heart disease through

the inhibition of cyclooxygenase and lipoxygenase activities in plateletsand macrophages.(Havsteen et al., 1983.)

The three flavonoids classes above are all ketone-containing compounds, and as such, are anthoxanthins (flavones and flavonols). This class was the first to be termed bioflavonoid. The terms flavonoids and bioflavonoid have also been more loosely used to describe non-ketone polyhydroxy polyphenol compounds which are more specifically termed flavonoids

Flavonoids (such as the catechins) are the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants.(spencer et al., 2008) Flavonols, the original bioflavonoid such as quercetin, are also found ubiquitously, but in lesser quantities. The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Foods with a high flavonoids content include parsley, onions, blueberries and other berries, black tea, green tea and oolong tea, bananas, all citrus fruits, red wine, and chocolate (with a cocoa content of 70% or greater).(Mc naught ., 1997).

1.2.9

Flavanone

CAROTENOIDS

Carotenoids are natural pigments which are synthesized by plants and are responsible for the bright colours of various fruits and vegetables.

Carotenoids generally cannot be manufactured by species in the animal kingdom so animals obtain carotenoids in their diets, and may employ them in various ways in metabolism.

There are over 600 known carotenoids; they are split into two classes, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, and contain no oxygen). All carotenoids are tetraterpenoids, meaning that they are produced from 8 isoprene molecules and contain 40 carbon atoms. In humans, three carotenoids (beta-carotene, alpha-carotene, and beta-cryptoxanthin) have vitamin A activity (meaning that they can be converted to retinal), and these and other carotenoids can also act as antioxidants. In the eye, certain other carotenoids (lutein, astaxanthin,(kidd., 2011) and zeaxanthin) apparently act directly to absorb damaging blue and near-ultraviolet light, in order to protect the macula of the retina, the part of the eye with the sharpest vision. Carotenoids with molecules containing oxygen, such as lutein and zeaxanthin, are known as xanthophylls. The unoxygenated (oxygen free) carotenoids such as α-carotene, β-carotene, and lycopene, are known as carotenes. Carotenes typically contain only carbon and hydrogen (i.e., are hydrocarbons), and are in the subclass of hydrocarbons. This class includes beta carotene, lutein and lycopene, which occur naturally in a number of fruits and vegetables, including carrots, spinach and tomatoes. They are known to help protect against some types of cancer and to strengthen the immune system.

1.3 GLUTATHIONE

Glutathione is a cysteine-containing peptide found in most forms of aerobic life. (Meister et al., 1998). It is not required in the diet and is instead synthesized in cells from its constituent acids. Glutathione has antioxidant properties since the thiols group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, glutathione is maintained in the reduced form by the enzyme glutathione reductase and in turn reduces other metabolites and enzyme systems, such as ascorbate in the glutathione-ascorbate cycle, glutathione peroxidases and glutaredoxins, as well as reacting directly with oxidants. (Wells et al., 1990).Due to its high concentration and its central role in maintaining the cell's redox state, glutathione is one of the most important cellular antioxidants. (Meister et al., 1998). In some organisms glutathione is replaced by other thiols, such as by mycothiol in the Actinomycetes, bacillithiol in some Gram-positive bacteria, or by trypanothione in the Kinetoplastids.(Fahey et al., 2001). Glutathione can directly neutralize ROS such as lipid peroxides, and also plays a major role in xenobiotic metabolism. Exposure of the liver to xenobiotic substances means the body prepares itself by increasing detoxification enzymes, i.e., cytochrome P-450 mixed-function oxidase. When an individual is exposed to high levels of xenobiotic, more glutathione is utilized for conjugation. Conjugation with Glutathione renders the toxin neutral and makes it less available to serve as an antioxidant.The glutathione system includes glutathione, glutathione reductase, glutathione peroxidases and glutathione ''S''-transferases. Of these glutathione peroxidase is an enzyme containing four selenium-cofactors that catalyzes the breakdown of hydrogen peroxide and organic hydroperoxides. Glutathione ''S''-transferases show high activity with lipid peroxides. These enzymes are at particularly high levels in the liver.

1.3.1 MELATONIN

Melatonin is a powerful antioxidant. Melatonin easily crosses cell membranes and the blood–brain barrier. (Reiter et al., 2009). Unlike other antioxidants, melatonin does not undergo redox cycling, which is the ability of a molecule to undergo repeated reduction and oxidation. Redox cycling may allow other antioxidants (such as vitamin C) to act as pro-oxidants and promote free radical formation. Melatonin, once oxidized, cannot be reduced to its former state because it forms several stable end-products upon reacting with free radicals. Therefore, it has been referred to as a terminal (or suicidal) antioxidant. (Tan et al., 2000). 

1.3.2 TOCOPHEROLS AND TOCOTRIENOLS (vitamin E)

Vitamin E is the collective name for a set of eight related Tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties.(Herrera et al., 2001) Of these, α-Tocopherols has been most studied as it has the highest bioavailability, with the body preferentially absorbing and metabolizing this form.(Brigelius-flohe et al., 1999). 

It has been claimed that the α-Tocopherols form is the most important lipid-soluble antioxidant, and that it protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction. This removes the free radical intermediates and prevents the propagation reaction from continuing. This reaction produces oxidized α-tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol. (Wang et al., 1999). This is in line with findings showing that α-Tocopherols, but not water-soluble antioxidants, efficiently protects glutathione peroxidases 4 (GPX4)-deficient cells from cell death. (Seiler et al., 2008) GPx4 is the only known enzyme that efficiently reduces lipid-hydroperoxides within biological membranes. The most important function of α-Tocopherols is as a signalling molecule. (Azzi et al., 2007) . γ-Tocopherols is a nucleophile that may react with electrophilic mutagens, and tocotrienols may be important in protecting neurons from damage.(S en et al., 2006).

1.3.3 SSUPEROXIDE DISMUTASES

Superoxide dismutases (SODs) are a class of closely related enzymes that catalyze the breakdown of the superoxide anion into oxygen and hydrogen peroxide.(Zelko et al.,2002) SOD enzymes are present in almost all aerobic cells and in extracellular fluids.(Johnson et al.,2005).superoxide dismutase enzymes contain metal ion cofactors that, depending on the isozyme, can be copper, zinc, manganese or iron. In humans, the copper/zinc SOD is present in the cytosol, while manganese SOD is present in the mitochondrion.( Bannister et al., 1987). There also exists a third form of SOD in extracellular fluids, which contains copper and zinc in its active sites.( Nozik-Grayck et al., 2005) .The mitochondrial isozyme seems to be the most biologically important of these three, since mice lacking this enzyme die soon after birth.(Melov et al., 1998). In contrast, the mice lacking copper/zinc SOD (Sod1) are viable but have numerous pathologies and a reduced lifespan while mice without the extracellular SOD have minimal defects (sensitive to hyperoxia).(Reaume et al., 1996 ). In plants, SOD isozyme are present in the cytosol and mitochondria, with an iron SOD found in chloroplasts that is absent from vertebrates and yeast.( Van Camp et al., 1997 ).

1.3.4 CATALASES

Catalases are enzymes that catalyse the conversion of hydrogen peroxide to water and oxygen, using either an iron or manganese cofactor.(Chelikani et al., 2004 ). Although hydrogen peroxide is its only substrate, it follows a ping-pong mechanism. Here, its cofactor is oxidised by one molecule of hydrogen peroxide and then regenerated by transferring the bound oxygen to a second molecule of substrate. (Hiner et al., 2002). 

1.3.5 PEROXIREDOXINS

Peroxiredoxins are peroxidases that catalyze the reduction of hydrogen peroxide, organic hydroperoxides, as well as peroxynitrite.( Rhee et al., 2005 ). They are divided into three classes: typical 2-cysteine peroxiredoxins; atypical 2-cysteine peroxiredoxins; and 1-cysteine peroxiredoxins.( Wood et al., 2003 ). These enzymes share the same basic catalytic mechanism, in which a redox- active cysteine (the peroxidatic cysteine) in the active site is oxidized to a sulfenic acid by the peroxide substrate.(Claiborne et al., 1999). Over-oxidation of this cysteine residue in peroxiredoxins inactivates these enzymes, but this can be reversed by the action of sulfiredoxin. (Johnson et al., 2007). Peroxiredoxins is important in antioxidant metabolism, as mice lacking peroxiredoxins 1 or 2 have shortened lifespan and suffer from haemolytic anaemia, while plants use peroxiredoxins to remove hydrogen peroxide generated in chloroplasts. (Dietz et al., 2006 ). 

1.3.6 THIOREDOXIN AND GLUTATHIONE SYSTEMS

The Thioredoxin system contains the 12-kDa protein thioredoxin and its companion thioredoxin reductase.(Nordberg et al., 2001).Proteins related to thioredoxin are present in all sequenced organisms. The active site of thioredoxin consists of two neighbouring cysteine, as part of a highly conserved CXXC motif, that can cycle between an active dithiol form (reduced) and an oxidized disulfide form. In its active state, thioredoxin acts as an efficient reducing agent, scavenging reactive oxygen species and maintaining other proteins in their reduced state. (Arnes et al., 2000). After being oxidized, the active thioredoxin is regenerated by the action of thioredoxin reductase, using NADPH as an electron donor. (Mustacich and Powis, 2000). The glutathione system includes glutathione, glutathione reductase, glutathione peroxidases and glutathione S-transferases.( Meister et al., 1983). This system is found in animals, plants and microorganisms.(Creissen et al., 1996).Glutathione peroxidase is an enzyme containing four selenium-cofactors that catalyzes the breakdown of hydrogen peroxide and organic hydroperoxides. There are at least four different glutathione peroxidase isozymes in animals.(Brigelius-Flohe., 1999), Glutathione peroxidase 1 is the most abundant and is a very efficient scavenger of hydrogen peroxide, while glutathione peroxidase 4 is most active with lipid hydroperoxides. Surprisingly, glutathione peroxidase 1 is dispensable, as mice lacking this enzyme have normal lifespans,( Ho Y et al., 1997) but they are hypersensitive to induced oxidative stress.(de Haan et al., 1998). In addition, the glutathione S-transferases show high activity with lipid peroxides.( Sharma et al., 2004 ). These enzymes are at particularly high levels in the liver and also serve in detoxification metabolism.( Hayes et al., 2005). 

1.3.7 LIPOIC ACID

This is another important endogenous antioxidant. It is categorized as “thiols” or “biothiol”. These are sulphur-containing molecules that catalyze the oxidative decarboxylation of alpha-keto acids, such as pyruvate and alphaketoglutarate, in the Krebs cycle.

Lipoic acid and its reduced form, dihydrolipoic acid (DHLA), neutralize the free radicals in both lipid and aqueous domains and as such has been called a universal antioxidant.

1.3.8 MEASUREMENT OF ANTIOXIDANTSMeasurement of antioxidants is not a straightforward process, as this is a diverse group of compounds with different reactivities to different reactive oxygen species. In food science, the oxygen radical absorbance capacity (ORAC) used to be the industry standard for antioxidant strength of whole foods, juices and food additives. (Cao et al., 1993).Consequently, the ORAC method, derived only in vitro experiments, is no longer considered relevant to human diets or biology.

Alternative in vitro measurements include the Folin-Ciocalteu reagent, and the Trolox equivalent antioxidant capacity assay. (Prior et al., 2005). Antioxidants are found in vegetables, fruits, grain cereals, eggs, meat, legumes and nuts. Some, such as lycopene and ascorbic acid, can be destroyed by long-term storage or prolonged cooking.( Rodriguez-Amaya ,2003). Other antioxidant compounds are more stable, such as the polyphenolic antioxidants in foods such as whole-wheat cereals and tea. (Baublis et al., 2000). Other antioxidants are not vitamins and are instead made in the body. For example, ubiquinol (coenzyme Q) is poorly absorbed from the gut and is made in humans through the mevalonate pathway.( Turunen et al., 2004). Another example is glutathione, which is made from amino acids.Although large amounts of sulphur-containing amino acids such as acetylcysteine can increase glutathione,(Dodd et al., 2008) .Supplying more of these precursors may be useful as part of the treatment of some diseases, such as acute respiratory distress syndrome, protein-energy malnutrition, or preventing the liver damage produced by paracetamol overdose.(Wu G et al., 2004). 

Other compounds in the diet can alter the levels of antioxidants by acting as pro-oxidants. Here, consuming the compound causes oxidative stress, which the body responds to by inducing higher levels of antioxidant defenses such as antioxidant enzymes.( Hail et al., 2008). 

There is great interest in determining their levels, the way they are related to pathological state. and they can be controlled by an antioxidant rich diet or by ingestion of antioxidant supplementation (Urquiaga and Leighton, 2000.,Croziet et al., 2000).In order to assess the capacity to remove the oxidative species of a given tissue, organ or physiological fluid, the concentration of a variety of antioxidants present in the medium must be determined. Due to their hydrophobicities, these will be distributed among the entire cellular compartment. A complete analysis of all the antioxidants is prevented by a large number of molecules that play this role, even in a single organelle.

oxidation reactions are crucial for life, they can also be damaging; plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, vitamin A, and vitamin E as well as enzymes such as catalases, superoxide dismutase and various peroxidases. Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. Oxidative stress is damage to cell structure and cell function by overly reactive oxygen-containing molecules and chronic excessive inflammation. Oxidative stress seems to play a significant role in many human diseases, including cancers. The use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. For these reasons, oxidative stress can be considered to be both the cause and the consequence of some diseases. Antioxidants are widely used in dietary supplements and have been investigated for the prevention of diseases such as cancer, coronary heart disease and even altitude sickness. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials with a limited number of antioxidants detected no benefit and even suggested that excess supplementation with certain putative antioxidants may be harmful. (Bailie et al., 2009).

1.3.9 MEDICINAL APPLICATIONS OF ANTIOXIDANTS

1.4 LANTHANIDES AS ANTI-CANCER AGENTS

The application of inorganic chemistry to medicine is a rapidly developing field, Novel therapeutics and diagnostic metal complexes are now having an impact on medical practice. Advances in bio-coordination chemistry are crucial for improving the design of compounds reduce toxic side effects and understand the mechanisms of action. A lot of metal –based drugs are widely used in the treatment of cancer (Xianquan et al.,2005). The clinical success of cisplatin and other platinum complexes is limited by significant side effects acquired or intrinsic resistance.Therefore, much attention has focused on designing new coordination compound with improved pharmacological properties and a

Broader range of antitumor activity (Blot et al., 1993). Strategies for developing new anticancer agents include the incorporation of carrier groups that can target tumor cells with high specificity. Also of interest is to develop complexes that bind to DNA in a fundamentally differentmanner than cisplatin, in an attempt to overcome the resistance pathway that has evolved to eliminate the drug. This review focuses on recent advancement in developing lanthanide coordination complexes.

1.4.1 LYCOPENE AS A POTENTIAL ANTICANCER AGENT

Dietary chemoprevention has emerged as a cost effective approach to control most prevalent chronic diseases including cancer. In particular, tomato and products are recognized to confer a wide range of health benefits. Epidemiology studies have provided evidence that high consumption of tomatoes effectively lowers the risk of reactive oxygen species (ROS)-mediated diseases such as cardiovascular diseases and cancer by improving the antioxidant carotenoid reported to be more stable and potent singlet oxygen quenching agent compared to other carotenoids. In addition to its antioxidants properties, lycopene shows an array of biological effects including cardio protective, antiinflammatory,anti-mutagenic and anti-carcinogenic activities. The cancer activities of lycopene have been demonstrated both in vitro and in vivo tumor models (Blotet al., 1993).

1.4.2 SELENIUM DERIVATIVES AS CANCER PREVENTIVE AGENTS

The role of selenium in the prevention of cancer has been recently established by laboratory experiments, clinical trials and epidemiological data. Consequently, selenium supplementation has moved from the realm of correcting nutritional deficiencies to one of pharmacological intervention, especially in the clinical domain of cancer chemoprevention and in the control of heart failure.Lipoic acid, the antioxidant’s antioxidant Lipoic acid protects against diseases of aging, this offerpowerful antioxidant protection against three common afflictions (two of them potentially disastrous) association with the aging stroke, heart attack and cataracts. It does it by suppressing the action of free radicals in the cells of the brain, heart and eyes. Lipid acid has an

Unusual relationship with four other important antioxidants:glutathione, coenzymeQ10, vitamin C and vitamin E.Memory loss is not considered to be not considered to be a disease at least not until it is a component of a fullfledged dementia, such as Alzheimer’s disease-but it iscertainly another hallmark of aging.Unlike lipoic acid other antioxidants are either primarily water-soluble or fat-soluble, but not both. This means that they have different (often overlapping) domain are free radical scavengers. What is good is that lipoic acid not only acts as a primary antioxidant in brain cells but serves to boost glutathione levels through the antioxidant network interactions.Diabetes, a terrible yet largely preventable diseaseis practically epidemic in the western world, especially the United States, because of our tendency to obesity due to poor diet and lack of exercise. Since lipoic acid is the most versatile and powerful antioxidant in the entireantioxidants defense network. Gene therapy promises to be one of the most exciting and fruitful avenues of medical practice in the twenty-first century and it offer powerful antioxidant protection against common afflictions.

1.5 CLASSIFICATION OF HIPPOCRATEA WELWITSCHII OLIV

Kingdom - plantae

Phylum - magnoliophyta

Class - magnoliopsida

Order - celastrales

Family - celastraceae

Genus - Hippocratea

Species - Hippocratea welwitschii.

1.5.1 COMMON NAMES

COMMON NAMES LANGUAGE AND ORIGIN

Bittersweet family English

Manogiegbini Liberia

Adangmeakladefi Ghana

Ijan Yoruba

Nyaworo uru ambombo Efik

Nyaworo uru ambombi Ibibio

(Burkill et al., 1985)

1.5.2 SYNONYMS: Simicratea welwitschii

1.5.3 DESCRIPTION

Hippocratea welwitschii (H.welwitschii) is a strong and extensive woody climber, wholly glabrous. Leaves are sub – membranous or at length coriaceous, broadly elliptical or obovate elliptical, rather obtusely cuspidate or shortly acuminate, reticulation scarcely prominent, 2 – 4 inches long.

1.5.4 DISTRIBUTION

The plant is commonly distributed in areas like: Angola, Cameroon, Congo, Gabon, Tanzania, Uganda, Togo Guinea, Benin, Ivory Coast and Nigeria.

1.5.5 PHYTOCHEMICAL ANALYSIS OF H. WELWITSCHII

The photochemical analysis showed that the root of the plant H. welwitschii Oliv contains saponins, alkaloids, phenols, terpenoids and glycosides in varying amounts – 1.66x10-2, 3.67x10-3, 2.64x10-2, 3.08x10-2 and 2.01x10-2 ug/g respectively. Minerals like potassium, sodium, chromium, calcium, magnesium, copper, manganese, iron, lead, zinc, Colbert, nikel, and selenium (Okoh – Esene et al., 2012).

1.5.6 ECONOMIC IMPORTANCE OF HIPPOCRATEA WELWITSCHII

Its phytochemical contents have attributed a great importance to the use of the plant in treatment of various diseases, the presence of saponins helps in binding blood cholesterol, thereby reducing heart problems but the most exciting and outstanding prospect for saponins is how they inhibit and kill cancer cells (Poornima and Ravishankar, 2009). It has also been reported that they do so without destroying normal cells in the process, as in the mode of some chemotherapeutic agents (Ryan and Shattu, 1994; Poornima and Ravishankar, 2009).

The presence of phenols, saponins and alkaloids in the root sample could confer antibiotic and hypoglycemic properties on the plant (Jacob and Burri, 1996). Phenols, saponins and alkaloids in the root of the plant are responsible for its use in the treatment of cough, dysentery, inflammations and ringworm (Frankel et al., 1993; Jacob and Burri, 1996).

The mineral composition of the root of Hippocratea welwitschii are known to play important metabolic and physiologic roles in the living system (Enechi and Odonwodo, 2003); Ujowundu et al., 2010). Iron, zinc, selenium are also known to prevent cardio – myopathy, muscle degeneration, growth retardation, alopecia, dermatitis, immunologic dysfunction, gonadal atrophy, impaired spermatogenesis, congenital malformations and bleeding disorders (Chaturvedi et al., 2004; Ujowundu et al., 2010).

Some traditional healers claimed that the root of the plant is used in the treatment of Malaria, Typhoid fever and Obesity. It was also claimed to stimulate menstruation (bark of the root), lower blood pressure, and used as a general body (internal) cleansing agent. It is prepared by boiling the bark of the root and taken as a drink or grinded into powdered form and taken with palpable food such as pap (akamu) or taken with tea without milk or sugar. Side effects only result when the extract is taken in excess, the common side effects are: drowsiness, dysentery and weakness of the body. However, it has not been shown if it can cure diabetes.


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