Agboola Victor

CHAPTER ONE 1.0 INTRODUCTION AND LITERATURE REVIEW 1.1 Introduction Today, oxidative stress has attracted the attention of researchers. An imbalance between free radicals and antioxidants leads to oxidative damage of proteins, fat, nucleic acids, and carbohydrates (Prior and Cao 1999). Oxidative stress can influence many biological processes such as apoptosis, viral proliferation, and inflammatory reactions. Inflammation is a local response (reaction) of living vascularized tissues to endogenous and exogenous stimuli. The term is derived from the Latin "inflammare" meaning to burn. Inflammation is fundamentally destined to localize and eliminate the causative agent and to limit tissue injury. However, chronic inflammation has been reported to be implicate in various diseases including cancer. Free radicals are central to the process of oxidative stress and chronic inflammations. Antioxidants have protected the body from the harmful effect of the free radicals (Azab et al., 2017). Endogenous antioxidants defense against the reactive oxygen species are strengthened by natural antioxidants that strengthen them and restore the optimal balance by neutralizing the ROS (Robinson et al., 2000). Medicinal plants are widely known to be a crucial source of natural antioxidants and anti-inflammatory agents (Aruoma, 2003). A medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical semisynthesis. When a plant is designated as medicinal, it is implied that the said plant is useful as a drug or therapeutic agent or an active ingredient of a medicinal preparation. Herbal medicines 1 are in great demand in the developed as well as in the developing countries for primary health care because of their wide biological and medicinal activities, higher safety margins and lesser costs (Yudharaj et al., 2016). Moringa oleifera is a plant that grows widely in many tropical and subtropical countries. It is known as the drumstick tree based on the appearance of its immature seed pods, the horseradish tree based on the taste of ground root preparations, and the ben oil tree from seed-derived oils. In some areas, immature seed pods are eaten, while the leaves are widely used as a basic food because of their high nutrition content (Thurber and Fahey, 2009; Mbikay, 2012; Razis et al., 2014). The leaves of Moringa oleifera are reported to contain various types of antioxidant compounds such as ascorbic acid, flavonoids, phenolics, and carotenoids (Alhakmani et al., 2013; Vongsak et al., 2014). According to several observations various preparations of Moringa oleifera are used for their anti-inflammatory, antihypertensive, diuretic, antihyperlipidemic, antineoplastic, antipyretic, antiulcer, cardioprotectant, and hepatoprotectant activities. Polysaccharides are polymeric carbohydrate molecules composed of long chains of monosaccharide units bound together by glycosidic bonds. Presence of polysaccharides is an indicator that Moringa oliefera could act as antiinflammatory, anti-clothing, anti-oxidant immune enhancers and hormone modulators (Akinmoladun et al., 2007), because polysaccharides have ability to block enzymes that cause inflammation, and can also modify prostaglandin pathways leading to protection of platelets from clumping (Ijeh et al., 2005). The present work was designed to access and compare antioxidantive and nitric oxide inhibitory activities of polysaccharides extract from Moringa oliefera leaves and seeds. 2 1.2 AIMS AND OBJECTIVES 1.2.1 Aim The aim of this work is to compare the anti-oxidative and nitric oxide inhibitory activities of polysaccharides extract from Moringa oleifera leaves and seeds. 1.2.2 Objectives The objectives of this research are to: i. Extract polysaccharides from Moringa oleifera leaves and seed using reported hot water method with slight modifications. ii. Assess and compare the hydrogen peroxide, nitric oxide, and hydroxyl radical scavenging activities of crude Water Soluble Polysaccharides extract from Moringa olefera leaves and seeds. iii. Evaluate and compare the Lipid peroxidation Inhibitory capacity iv. Assess and compare the total antioxidant activities. 3 1.3 LITERATURE REVIEW 1.3.1 Oxidative Stress The close association between oxidative stress and lifestyle-related diseases has become well known. Oxidative stress is defined as a “state in which oxidation exceeds the antioxidant systems in the body secondary to a loss of the balance between them.” It not only causes hazardous events such as lipid peroxidation and oxidative DNA damage, but also physiologic adaptation phenomena and regulation of intracellular signal transduction. From a clinical standpoint, if biomarkers that reflect the extent of oxidative stress were available, such markers would be useful for physicians to gain an insight into the pathological features of various diseases and assess the efficacy of drugs (Toshikazu and Yuji, 2002). 1.3.2 Biomarkers of Oxidative Stress The biomarkers that can be used to assess oxidative stress have been attracting interest because the accurate assessment of such stress is necessary for investigation of various pathological conditions, as well as to evaluate the efficacy of drugs. Assessment of the extent of oxidative stress using biomarkers is interesting from a clinical standpoint. The markers found in blood, urine, and other biological fluids may provide information of diagnostic value, but it would be ideal if organs and tissues suffering from oxidative stress could be imaged in a manner similar to CT scanning and MR imaging. In recent years, attempts have been made to use electron spin resonance techniques for this purpose, but it will take time before such methods can be applied to humans. Because the body is not necessarily fully protected against oxidative damage, some of its constituents may be injured by free radicals, and the resultant oxidative products have usually 4 been used as markers. Many markers have been proposed, including lipid peroxides, malondialdehyde, and 4-hydroxynonenal as markers for oxidative damage to lipids; isoprostan as a product of the free radical oxidation of arachidonic acid; 8-oxoguanine (8-hydroxyguanine) and thymineglycol as indicators of oxidative damage to DNA; and various products of the oxidation of protein and amino acids including carbonyl protein, hydroxyleucine, hydrovaline, and nitrotyrosine. Lipid peroxide was assessed in clinical samples even in relatively early studies, and the analytical methods for this substance have improved (Toshikazu and Yuji, 2002). 1.3.3 Oxidative Stress as a Biological Modulator and as a Signal Oxidative stress not only has a cytotoxic effect, but also plays an important role in the modulation of messengers that regulate essential cell membrane functions, which are vital for survival. It affects the intracellular redox status, leading to the activation of protein kinases, including a series of receptor and non-receptor tyrosine kinases, protein kinase C, and the MAP kinase cascade, and hence induces various cellular responses. These protein kinases play an important role in cellular responses such as activation, proliferation, and differentiation, as well as various other functions. Accordingly, the protein kinases have attracted the most attention in the investigation of the association between oxidative stress and disease (Toshikazu and Yuji, 2002). Oxidative stress can influence many biological processes such as apoptosis, viral proliferation, and inflammatory reactions. In these processes, gene transcription factors such as nuclear factor and activator protein-1 (AP-1) act as oxidative stress sensors through their own oxidation and reduction cycling. This type of chemical modification of proteins by oxidation and reduction is called reduction-oxidation (redox) regulation (Toshikazu and Yuji, 2002). 5 1.4 Inflammation Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants (Ferrero-Miliani et al., 2007) and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair. The five cardinal signs are heat, pain, redness, swelling, and loss of function (Ferrero-Miliani et al., 2007). Inflammation is a generic response, and therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity, which is specific for each pathogen (Abbas and Lichtman, 2009). Too little inflammation could lead to progressive tissue destruction by the harmful stimulus (e.g. bacteria) and compromise the survival of the organism. In contrast, too much inflammation, in the form of chronic inflammation, is associated with various diseases, such as hay fever, periodontal disease, atherosclerosis, and osteoarthritis. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli, and is achieved by the increased movement of plasma and leukocytes (in particular granulocytes) from the blood into the injured tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation, such as mononuclear cells, and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Inflammation has also been classified as Type 1 and Type 2 based on the type of cytokines and helper T cells (Th1 and Th2) involved (Berger, 2000). 6 1.4.1 Acute Inflammation Acute inflammation occurs immediately upon injury, lasting only a few days (Hannoodee and Nasuruddin, 2020). Cytokines and chemokines promote the migration of neutrophils and macrophages to the site of inflammation (Hannoodee and Nasuruddin, 2020). Pathogens, allergens, toxins, burns, and frostbite are some of the typical causes of acute inflammation. Tolllike receptors (TLRs) recognize microbial pathogens. Acute inflammation can be a defensive mechanism to protect tissues against injury. Inflammation lasting 2–6 weeks is designated subacute inflammation (Pahwa et al., 2020). Acute inflammation is a short-term process, usually appearing within a few minutes or hours and begins to cease upon the removal of the injurious stimulus (Robbins et al., 1998). It involves a coordinated and systemic mobilization response locally of various immune, endocrine and neurological mediators of acute inflammation. In a normal healthy response, it becomes activated, clears the pathogen and begins a repair process and then ceases (Kumar et al., 2004). It is characterized by five cardinal signs: (Chandrasoma and Taylor, 2005). i. Redness ii. Swelling iii. Heat iv. Pain or discomfort v. Loss of function. (Werner, 2009). 1.4.2 Chronic Inflammation Chronic inflammation is inflammation that lasts for months or years. Macrophages, lymphocytes, and plasma cells predominate in chronic inflammation, in contrast to the neutrophils that 7 predominate in acute inflammation (Pahwa et al., 2020). Diabetes, cardiovascular disease, allergies, and chronic obstructive pulmonary disease (COPD) are examples of diseases mediated by chronic inflammation (Pahwa et al., 2020). Obesity, smoking, stress, insufficient diet and poor diet are some of the factors that promote chronic inflammation. 1.4.3 Causes of Inflammation The principal cause of inflammation is a pure mechanical pressure, including blunt trauma (Cohen et al., 2009), foreign bodies (Subramanian 2003), vibrations (Abi-Hachem 2010) and chronic pressure of low intensity (Davis and Handy 1996). The basic mechanism of causing inflammation by pressure is most probably through tissue hypoxia. Namely, tissue oxygen as a liposoluble substance is distributed mainly in lipids and hydrophobic proteinaceous matter.  Exogenous  Mechanical  Physical  Chemical  Biological  Endogenous  Circulatory disorder, hypoxia  Endogenous protease release  Immuncomplex formation 1.4.4 The Classic Signs of Inflammation  Redness  Swelling tumor 8 1.5  Heat  Pain or discomfort  Loss of function (Ferrero-Miliani et al., 2007). MEDICINAL PLANT The term of medicinal plants include a various types of plants used in herbalism and some of these plants have a medicinal activities. These medicinal plants consider as a rich resources of ingredients which can be used in drug development and synthesis. Besides that these plants play a critical role in the development of human cultures around the whole world. Moreover, some plants consider as important source of nutrition and as a result of that these plants recommended for their therapeutic values. These plants include ginger, green tea, walnuts and some others plants. Other plants their derivatives consider as important source for active ingredients which are used in aspirin and toothpastes (Bassam, 2012) has been estimated that about 13,000 species of plants have been employed for at least a century as traditional medicines by various cultures around the world. A list of over 20,000 medicinal plants has been published, and very likely a much larger number of plants. The medicinal value of plants has assumed a more important dimension in owing largely to the discovery that extract from plant contain not only minerals and primary metabolites but also diverse array of secondary metabolites with antioxidant potential. Antioxidant substances block the action of free radicals which have been implicated in the pathogenesis of the diseases such as neurodegenerative disease, Alzheimer’s diseases etc. (Aruoma, 2003). 9 1.5.1 Characteristics of Medicinal Plants Synergic medicine- The ingredients of plants all interact simultaneously, so their uses can complement or damage others or neutralize their possible negative effects Support of Official Medicine- in the treatment of complex cases like cancer diseases the components of the plants proved to be very effective. Preventive Medicine- It has been proven that the component of the plants also characterize by their ability to prevent the appearance of some diseases. This will help to reduce the use of the chemical remedies which will be used when the disease is already present i.e., reduce the side effect of synthetic treatment. 1.5.2 Classification of Medicinal Plants Classification of medicinal plants is organized in different ways depending on the criteria used. In general, medicinal plants are arranged according to their active principles in their storage organs of plants, particularly roots, leaves, flowers, seeds and other parts of plant. These principles are valuable to mankind in the treatment of diseases. Reports on the classification of many plant species yielding vegetable oils used in cosmetics and body and skin care preparations are sporadic or lacking (Yudharaj et al.,- Classification According to the Usage The herbs are classified in four parts: medicinal herbs, culinary herbs, aromatic herbs, ornamental herbs.  Medicinal Herbs have curative powers and are used in making medicines because of their healing properties like marigold, lemon balm, lavender, johnny-jump-up, feverfew etc. 10  Culinary Herbs are probably the mostly used as cooking herbs because of their strong flavours like oregano, parsley, sweet basil, horseradish, thyme etc. (Krishnaiah et al, 2011).  Aromatic Herbs have some common uses because of their pleasant smelling flowers or foliage. Oils from aromatic herbs can be used to produce perfumes, toilet water, and various scents. For e.g. mint, rosemary, basil etc. (Krishnaiah et al, 2011).  Ornamental Herbs are used for decoration because they have brightly colored flowers and foliage like lavender, chives, bee balm, lemongrass etc. (Krishnaiah et al, 2011). 1.5.4 Classification according to the Active Constituents According to the active constituents all herbs are divided into five major categories: Aromatic (volatile oils), Astringents (tannins), Bitter (phenol compounds, saponins, and alkaloids), Mucilaginous (polysaccharides), and Nutritive (food stuffs). Aromatic herbs The name is a reflection of the pleasant odour that many of these herbs have. They are used extensively both therapeutically and as flavourings and perfumes. Aromatic herbs are divided into two subcategories: stimulants and nervines. Stimulant Herbs increase energy and activities of the body, or its parts or organs, and most often affect the respiratory, digestive, and circulatory systems. E.g. fennel, ginger, garlic, lemongrass. Astringent Herbs Tannins in Astringent Herbs have the ability to precipitate proteins, and this "tightens," contracts, or tones living tissue, and helps to halt discharges. They affect the digestive, urinary, and 11 circulatory systems, and large doses are toxic to the liver. They are analgesic, antiseptic, ant abortive, astringent, emmenagogue, hemostatic, and styptic (Shree Devi, 2011). Bitter Herbs Bitter Herbs are named because of the presence of phenols and phenol glycosides, alkaloids, saponins and are divided into four subcategories: Diuretic Herbs induce loss of fluid from the body through the urinary system. The fluids released help cleanse the vascular system, kidneys, and liver. They are alterative, antibiotic, ant catarrhal, antipyretic, and antiseptic, lithotripter, and blood purifier in nature. asparagus, blessed thistle, burdock, butcher's broom, buchu, chaparral, chickweed, corn silk, dandelion, dog grass, grapevine, and parsley (Shree Devi, 2011). Mucilaginous Herbs Mucilaginous herbs derive their properties from the polysaccharides they contain, which give these herbs a slippery, mild taste that is sweet in water. All plants produce mucilage in some form to store water and glucide as a food reserve. They eliminate the toxins from the intestinal system, help in regulating it and reduce the bowel transit time. They are antibiotic, antacid,demulcent, emollient, vulnerary, and detoxifier in nature. For e.g. althea, aloe, burdock, comfrey,dandelion, Echinacea, fenugreek, kelp, psylium,slippery elm, dulse, glucomannan from Konjak root, Irish moss, and mullein (Shree Devi, 2011). Nutritive Herbs Wheat germ these herbs derive both their name and their classification from the nutritive value they provide to the diet. They are true foods and provide some medicinal effects as fibber, mucilage, and diuretic action. But most importantly they provide the nutrition of protein, 12 carbohydrates, and fats, plus the vitamins and minerals that are necessary for adequate nutrition. For e.g. rosehips, acerola, apple, asparagus, banana, barley grass, bee pollen,bilberry, broccoli, cabbage, carrot, cauliflower, grapefruit, hibiscus, lemon, oat straw, onion, orange, papaya, pineapple, red clover, spirulina, stevia. 1.6 Moringa oleifera Moringa oleifera is a tree that grows widely in many tropical and subtropical countries. It is grown commercially in India, Africa, South and Central America, Mexico, Hawaii, and throughout Asia and Southeast Asia. It is known as the drumstick tree based on the appearance of its immature seed pods, the horseradish tree based on the taste of ground root preparations, and the ben oil tree from seed-derived oils. In some areas, immature seed pods are eaten, while the leaves are widely used as a basic food because of their high nutrition content (Thurber and Fahey, 2009; Mbikay, 2012; Razis et al., 2014). No human clinical trials have been conducted looking at the efficacy of M. oleifera for treating under nutrition. Seeds, leaves, oil, sap, bark, roots, and flowers are widely used in traditional medicine. Moringa leaves have been characterized to contain a desirable nutritional balance, containing vitamins, minerals, amino acids, and fatty acids (Moyo et al., 2011; Teixeira et al., 2014; Razis et al., 2014). Additionally, the leaves are reported to contain various types of antioxidant compounds such as ascorbic acid, flavonoids, phenolics, and carotenoids (Alhakmani et al., 2013; Vongsak et al., 2014). According to several commentaries (Anwar et al., 2007; Mbikay, 2012; Razis et al., 2014), various preparations of M. oleifera are used for their anti-inflammatory, antihypertensive, diuretic, antihyperlipidemic, antineoplastic, antipyretic, antiulcer, cardioprotectant, and hepatoprotectant activities. The therapeutic potential of M. oleifera leaves in treating hyperglycemia and dyslipidemia was reviewed by (Mbikay 2012). (Razis et al., 2014) 13 summarized potential health benefits of M. oleifera, focusing on their nutritional content as well as antioxidant and anti-inflammatory characteristics. Figure 1.1: Showing the Leave of Moringa Oleifera (Wikipedia) . Figure 1.2: Showing the Seed of Moringa Oleifera (Wikipedia) 14 1.6.1 Taxonomy Table 1.1: Scientific Classification of Moringa Oleifera (Wikipedia) Kingdom: Plantea Clade: Tracheophytes Clade: Angiosperm Clade: Eudicots Clade: Rosids Order: Brassicales Family: Moringaceae Genus: Moringa Species: M. oleifera 1.6.2 Description Moringa oleifera is a fast-growing, deciduous tree (horseradish tree encycloprdia Britannica, 2015) that can reach a height of 10–12 m (32–40 ft) and trunk diameter of 45 cm (1.5 ft) (Parotta and John, 1993). The bark has a whitish-grey color and is surrounded by thick cork. Young shoots have purplish or greenish-white, hairy bark. The tree has an open crown of drooping, fragile branches, and the leaves build up feathery foliage of tripinnate leaves. The flowers are fragrant and hermaphroditic, surrounded by five unequal, thinly veined, yellowish-white petals. The flowers are about 1.0–1.5 cm (1/2 in) long and 2.0 cm (3/4 in) broad. They grow on slender, hairy stalks in spreading or drooping flower clusters, which have a length of 10–25 cm (Parotta and John, 1993). 15 Flowering begins within the first six months after planting. In seasonally cool regions, flowering only occurs once a year in late Spring and early Summer (northern hemisphere between April and June, southern hemisphere between October and December). In more constant seasonal temperatures and with constant rainfall, flowering can happen twice or even all year-round (Parotta and John, 1993). The fruit is a hanging, three-sided brown capsule of 20–45 cm size, which holds dark brown, globular seeds with a diameter around 1 cm. The seeds have three whitish papery wings and are dispersed by wind and water (Parotta and John, 1993). In cultivation, it is often cut back annually to 1–2 m (3–6 ft) and allowed to regrow so the pods and leaves remain within arm's reach (Parotta and John, 1993). 1.6.3 Medicinal Properties of Moringa oleifera The Moringa’s incredible medicinal usage which is claimed by many cultures and communities based on real-life experiences are now slowly being confirmed by science. Though research, the moringa was found to contain many essential nutrients, for instance, vitamins, minerals, amino acids, beta-carotene, oxidants, anti-inflammatory nutrients and omega 3 and 6 fatty acids (Fahey, 2005; Hsu et al., 2006; Kasolo et al., 2010). Nutrition content of a plant plays an essential; function in medicinal, nutritional and therapeutic properties (Al-kharusi et al., 2009). It is believed that moringa leave to consist high source of vitamin C, calcium, β-carotene, potassium as well as protein. It works as an effective source of natural antioxidants. Due to the presence of several sorts of antioxidants compounds such flavonoids, ascorbic acids, carotenoids, and phenolic, moringa is able to extend the period of food contaminating fats (Dillard and German, 2000; Siddhuraju and Becker, 2003). 16 It was reported that several researchers at the Asian vegetables research and development Centre (AVRDC) that the leaves of four of the Moringa species were rich in nutrients and antioxidants (price, 2007) in which the nutrients content varies with a few factors such as preparation method, leaf age and harvest season. In addition, the Moringa was found to have a group of unique compounds containing sugar and rhamnose, which are uncommon sugar-modified glucosinolates (Fahey et al., 2001; Fahey, 2005; Amaglo et al., 2010). These compounds were reported to demonstrate certain chemopreventive activity, by inducing apoptosis (Brunelli et al., 2010). 1.6.4 Antioxidant Activity of Moringa oleifara Leaf Naturally, occurring antioxidants, particularly polyphenols, are the main plant compounds that are able to decrease oxidative damage in tissues by indirect enhancement of a cell or by free radical; scavenging (Du et al., 2010). The leaves of the Moringa oleifera tree have been reported to demonstrate anti-oxidative activity dues to its high amount of polyphenols (Sreelatha and Padma, 2009; Verma et al., 2009). Moringa oleifera extract of both mature and tender leaves exhibit strong antioxidant activity against free radicals, prevent oxidative damage to major biomolecules and gives significant protection against oxidative damage (Sreelatha and Padma, 2009). A comparative study indicates that mature Moringa oleifera leaf extract exhibited better values of enzymatic and non-enzymatic antioxidants. In the DPPH (2,2-Diphenyl-1-Picrylhydrazyl) free radical scavenging activity test, both mature and tender leaf extract showed significant reduction of DPPH radicals. The scavenging activity was suggested to be attributed to its hydrogen donating ability and was seen more in the mature leaf extract (Sreelatha and Padma, 2009). 17 The antioxidant properties of the Moringa oleifera was also examined but (Verma et al., 2012) whereby 50% ethanolic leaf extract was tested to study the lipid peroxidation (LPO), catalase (CAT) and superoxide dismutase (SOD) activities. The antioxidant properties of the Moringa extract was found to change in SOD, CAT and LPO levels inn rat gastric mucosa. There was a reported increase in gastric mucosa SOD and LPO activates during ulcer conditions which indicated an antioxidant defense mechanism by the Moringa oleifera extract (Verma et al., 2012). 1.6.5 Anti-Inflammatory Activity of Moringa oleifera Leaf Moringa oleifera leaf has been practically used in medicinal field, throughout the decades to heal a huge amount of acute and chronic conditions. In vitro and in vivo studies with the plant have recommended its effectiveness in treating inflammation (Bennett et al., 2003; Fahey, 2005; Mbikay, 2012). The properties of its phytochemicals, such as flavonoids and phenolic acids were related to the anti-inflammaotry activites (Mbikay, 2012). It has been discovered that hepatic myeloperoxidase activity can be carried out as a marker of inflammation and tissue neurophill accumulation and activation (Hillefass et al., 1990). 1.6.6 Anti-Inflammatory Activities of Moringa oleifera Seed In a study by (Caceres et al., 1992), infused Moringa oleifera seeds showed inhibition of carrageenan-induced hind paw edema. The inhibition by the seed infusion conversely was dosedependent as compared to the root infusion which showed inactivity of more convincing antiinflammatory inhibition. The hepatoprotective properties of Moringa seed extract which was discovered from the antifibrotic study of (Hamza, 2010) indicated that the moringa also possessed anti-inflammatory 18 properties against CC14-induced liver damage and fibrosis. This finding was confirmed by the decrease of globulin level in serum and the myeloperoxidase activity in liver. Additionally, in the histopathological analysis, a decrease in inflammatory cells infiltrations was discovered. 1.7 ANTIOXIDANT Antioxidants are substances that may protect cells from the damage caused by unstable molecules known as free radicals. Antioxidants interact with and stabilize free radicals and may prevent some of the damage free radicals might otherwise cause. Free radical damage may lead to cancer. Examples of antioxidants include beta-carotene, lycopene, vitamins C, E, A and other substances (Sies, 1997). An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols (Sies, 1997). 1.7.1 Mode of Action of Antioxidant Antioxidant acts by:  Breaking chain reaction e.g. tocopherol  Reducing the concentration of reactive oxygen species e.g. glutathione  Scavenging initiating radicals  Chelating the transition metal catalyst 19 1.7.2 Classification of Antioxidants Antioxidants are grouped into two namely;  Primary or natural antioxidants.  Secondary or synthetic antioxidants Primary or Natural Antioxidants They are the chain breaking antioxidants which react with lipid radicals and convert them into more stable products. Antioxidants of this group are mainly phenolic in structures and include the following (Hurrell, 2003): Antioxidants minerals - These are co factor of antioxidants enzymes. Their absence will definitely affect metabolism of many macromolecules such as carbohydrates. Examples include selenium, copper, iron, zinc and manganese. Antioxidants vitamins – It is needed for most body metabolic functions. They include-vitamin C, vitamin E, vitamin B. Phytochemicals - These are phenolic compounds that are neither vitamins nor minerals. These include: Flavonoids: These are phenolic compounds that give vegetables fruits, grains, seeds leaves, flowers and bark their colors. Catechins are the most active antioxidants in green and black tea and sesamol. Carotenoids are fat soluble color in fruits and vegetables. Beta carotene, which is rich in carrot and converted to vitamin A when the body lacks enough of the vitamin. Lycopene, high in tomatoes and zeaxantin is high in spinach and other dark greens. Herbs and spices-source 20 include Diterpene, rosmariquinone, thyme, nutmeg, clove, black pepper, ginger, garlic and curcumin and derivatives. Secondary or Synthetic Antioxidants These are phenolic compounds that perform the function of capturing free radicals and stopping the chain reactions, the compounds include (Hurrell, 2003):  Butylated hydroxyl anisole (BHA).  Butylated hydroxyrotoluene (BHT).  Propyl gallate (PG) and metal chelating agent (EDTA).  Tertiary butyl hydroquinone (TBHQ).  Nordihydro guaretic acid (NDGA). 1.7.3 Various Type Antioxidants In present time various antioxidant found in food viz. natural antioxidants, synthetic antioxidants, dietary antioxidant, endogenous antioxidant which play a important role in preservation of food. Dietary Antioxidants: The dietary antioxidants such as ascorbates, tocopherols and carotenoids are well known and there is a surplus of publications related to their role in health (Boskou et al., 2005). Vitamin C, vitamin E, and beta carotene, Beta carotene and other carotenoids and oxycarotenoids, e.g., lycopene and luteinare among the most widely studied dietary antioxidants. In extracellular fluids vitamin C is considered the most important water-soluble antioxidant. It is capable of neutralizing ROS in the aqueous phase before lipid peroxidation is initiated. Vitamin E, a major lipid-soluble antioxidant, is the most effective chain-breaking antioxidant within the cell membrane where it protects membrane fatty acids from lipid peroxidation. Vitamin C has 21 been cited as being capable of regenerating vitamin E (Sies, 1992). Beta carotene and other carotenoids are also believed to provide antioxidant protection to lipid-rich tissues. Research suggests beta carotene may work synergistically with vitamin E (Jocab, 1995). 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 anticarcinogenic activity (Cody et al., 1986; Kuhnau et al., 1976; Havsteen, 1983 and Middleton, 1984). Synthetic Antioxidant: Synthetic antioxidants are chemically synthesized since they do not occur in nature and are added to food as preservatives to help prevent lipid oxidation (Shahidi et al., 1992). These antioxidants fall into two major categories depending on their mode of action Primary antioxidants and Secondary antioxidants. The primary antioxidants, which prevent the formation of free radicals during oxidation, can further include three major categories. Natural Antioxidant: Natural antioxidants are constituents of many fruits and vegetables and they have attracted a great deal of public and scientific attention (Diwani et al., 2009). Natural antioxidants occur in all parts of plants. Food tissues, because they are (or were) living, are under constant oxidative stress from free radicals, reactive oxygen species, and prooxidants generated both exogenously (heat and light) and endogenously (H2O2 and transition metals). For this reason, many of these tissues have developed antioxidant systems to control free radicals, lipid oxidation catalysts, oxidation intermediates, and secondary breakdown products (Chen, 2008). Endogenous Antioxidants: In addition to dietary antioxidants, the body relies on several endogenous defense mechanisms to help protect against free radical-induced cell damage. The antioxidant enzymes – glutathione peroxidase, catalase, and superoxide dismutase (SOD) – 22 metabolize oxidative toxic intermediates and require micronutrient cofactors such as selenium, iron, copper, zinc, and manganese for optimum catalytic activity. It has been suggested that an inadequate dietary intake of these trace minerals may compromise the effectiveness of these antioxidant defense mechanisms (Duthie and Brown, 1994). Glutathione, an important watersoluble antioxidant, is synthesized from the amino acids glycine, glutamate, and cysteine. Glutathione directly quenches ROS such as lipid peroxides, and also plays a major role in xenobiotic metabolism. Exposure of the liver to xenobiotic substances induces oxidative reactions through the upregulation of detoxification enzymes, i.e., cytochrome P-450 mixed function oxidase. When an individual is exposed to high levels of xenobiotics, more glutathione is utilized for conjugation (a key step in the body’s detoxification process) making it less available to serve as an antioxidant. Research suggests that glutathione and vitamin C work interactively to quench free radicals and that they have a sparing effect upon each other (Jocab, 1995). Lipoic acid, yet another important endogenous antioxidant, categorized as a “thiol” or “biothiol,” is a sulfur-containing molecule that is known for its involvement in the reaction that catalyzes the oxidative decarboxylation of alpha-keto acids, such as pyruvate and alphaketoglutarate, in the Krebs cycle. Lipoic acid may also exert its antioxidant effect by chelating with prooxidant metals. Research further suggests that lipoic acid has a sparing effect on other antioxidants (Kagen, 1992). Exogenous: Exogenous antioxidants can derive from natural sources (vitamins, flavonoids, anthocyanins, some mineral compounds), but can also be synthetic compounds, like butylhydroxyanisole, butylhydroxytoluene, gallates, etc. (Litescu et al., 2011). There is an increasing interest in antioxidants, particularly in those intended to prevent the presumed 23 deleterious effects of free radicals in the human body, as well as the deterioration of fats and other constituents of foodstuffs (Molyneux, 2004). 1.7.4 Characteristics of Antioxidants The major antioxidants currently used in foods are monohydroxy or polyhydroxy phenol compounds with various ring substitutions. These compounds have low activation energy to donate hydrogen. Hence, the resulting antioxidants radical does not initiate another free radical due to the stabilization of the delocalized radical electron. Propagation and initiation of free radicals chain reaction can be delayed or minimized by the donation of hydrogen from the antioxidants and metal chelating agent. The resulting antioxidant free-radical is not subject to rapid oxidation due to its stability. Antioxidants free-radicals can also react with lipid free radicals to form a stable complex compound thereby preventing some of their damages. 1.8 Polysaccharide Polysaccharides (glycans) are long chains of monosaccharides. Each monosaccharide is connected together via glycosidic bonds to form the polymeric structure known as polysaccharide. Polysaccharides are the largest component of biomass. It is estimated that more than 90% of the carbohydrate mass in nature is in the form of polysaccharides (Lehninger Principles of Biochemistry). 1.8.1 Generalised Functions of Polysaccharides 1. Polysaccharides plays vital role in energy storage and acts as cellular fuel source such as glycogen and starch. 24 2. Polysaccharides help to maintain the structural integrity of the organisms such as cellulose helps to maintain structure in plants and chitin is chief component of animal exoskeleton. 3. Polysaccharides are also present in extracellular space such as in animal tissue that helps in maintain shape, supports cells, tissue and organ. 4. Polysaccharides are an integral part of cell to cell communication and cellular recognition (Lehninger Principles of Biochemistry). 1.8.2 Applications of Polysaccharides 1. Uses as Surface-Acting Drugs and in Medicinal Formulations: Polysaccharides have long been used in pharmaceuticals and as surface-acting drugs. Their utility in medicinal formulations depends upon the extraordinary range and combination of useful properties that can be found among them and their simple derivatives (Pandey and Chowdhury, 2000). Among the materials approved for pharmaceutical use are U.S.P. grades of acacia (gum arabic); tragacanth (gum tragacanth); agar; glycyrrhiza; sodium and calcium alginate; starch; and methyl; carboxymethyl, and hydroxypropyl cellulose. Typically, these are used as suspending and emulsifying agents, tabletting aids, viscosity-control aids, and coating materials (Poyton, 2009). Carboxymethylcellulose has been used as a tablet-coating material for enteric treatments since it is insoluble in stomach acid and soluble at alkaline pHs. The water-soluble cellulose derivatives are also used in contact-lens solutions and ophthalmic preparations. Alginates are used as dentalimpression materials with di- or trivalent metal salts as gelling agents (Rabelo et al., 2003). 2. Uses in Blood, Body Fluids, and Biomateriais, and in Trauma: In the applications discussed above, the polymers cannot readily cross the skin or mucous-membrane barrier and 25 enter the body proper, so many synthetic and natural polymers may be safely used. For the following uses, polysaccharides are introduced into the organism and the requirements for safe and effective use are far more stringent (Pessoa et al., 2002). In general, the substance should remain within the tissue or the general circulation for a period of time adequate to perform its desired function and yet should be eventually excreted or metabolized. This requirement is more readily met by many polysaccharides than by most synthetic polymers so far tested. A more important function is that of plasma expander, and dextran is considered to possess nearly ideal attributes and has been used successfully in the treatment of problems associated with loss of whole blood or plasma (Silva et al., 2005). 26 CHAPTER TWO 2.0 MATERIALS AND METHOD 2.1 Chemicals and apparatus used Ethanol, Hydrogen peroxide, Ferric sulfate, ethanolic salicyclic acid, phenol, sulfuric acid, Glucose, NaCl, Ibuprofen, Bovine Serum Albumin, Diclofenac, and distilled water. The apparatus used for the experiment include Oven, 80 mesh size sieve, Airtight container, Centrifuge, Spectrophotometer, Dessicator, Dual beam UV spec, Anti-coagulant bottle (3.8% w/v tri-sodium citrate), Ice bucket, Refridgerator, Clean and dried test tubes 2.2 Plants Collection and Authentication Fresh leaves of Moringa oleifera were gotten from Sasa Market, Oke Baale, Osogbo, Osun State, Nigeria while fresh seeds of Moringa Oleifera were gotten from MFMC fellowship Centre Uniosun school road, Osogbo, Osun State, Nigeria and were identified and authenticated in the Herbarium of the Forestry Research Institute of Nigeria (FRIN). 2.2.1 Plant Treatment and Preparation The fresh leaves of Moringa oleifera were oven dried at -- temperature for two days, the leaves were grinded to powdery form using a manual blender and mortar and pestle. The Moringa oleifera seeds were Air-dried for two days at normal temperature and were grinded to powdery form with mortar and pestle. The 30g of the powdered samples were weighed and mixed with 200 ml of distilled water at 65˚C under stirred condition for 3hrs. Then centrifuged the extracts at 3,000 rpm for 30 min and three volume of 99% ethanol was added to the supernatant in order to precipitate the polysaccharide and kept at 4˚C overnight. The precipitate was then freeze-dried to 27 extract the crude polysaccharide fraction. Then the crude were stored in refrigerator for further use. 2.3 In Vitro Antioxidant Assays 2.3.1 Hydrogen Peroxide Radical Scavenging Assay H2O2 scavenging activity of the plant polysaccharides (PPS) was determined based on (Ruch et al.. 1989). 2 ml of 40mM H2O2 solution prepared in 0.1 M phosphate buffer pH 7.4 was added to 1ml of various concentrations of the sample and incubated for 10 min at room temperature. The absorbance was measured at 374 nm using phosphate buffer as a blank solution. Ascorbic acid was used as a standard. The percentage of scavenging activity was calculated as: Scavenging activity (%) = 1- A sample / A control X 100 2.3.2 Hydroxyl Radical Scavenging Assay The HO- scavenging ability of the PPS was calculated by a Fenton-type reaction (Chen et al., 2016). 1ml of different concentrations of the PPS was mixed with 1 ml of 9mM ferric sulfate, 50ml of 9mM ethanolic salicylic acid and 50ml of 9mM hydrogen peroxide. The mixture was incubated for 30min at 37˚C. The absorbance was measured at 490 nm. Ascorbic acid was used as a positive control. The activity was calculated as above. 28 2.3.3 Total Antioxidant Assay The antioxidant capabilities of the plant sample were assured by phosphomolybdenum assay as described in the methodology of Umamaheswari and Chatterjee (2008). Phosphomolybdenum reagent solution was prepared by mixing Na3PO4 (28mM) and H2SO4 (0.6M) with that of ammonium molybdate (4mM). The reaction mixture is heated at 95 °C in water bath for 90min taking a good care that it is fully covered with silver foil to avoid direct light exposure. After this heat treatment, the reaction mixture was cooled at room temperature for some time and submitted to spectrophotometric analysis at765 nm. Ascorbic acid serves as a standard in this assay. 2.3.4 Lipid Peroxidation Inhibitory Assay The lipid peroxidation inhibition assay (LPI) was determined according to the method described by Liu and Ng (2003). 100μl 10mM FeSO4, 100μl 0.1mM Ascorbic acid and 0.3ml of extractives or standard at different concentration were mixed to make the final Volume 1 ml. The reaction mixture was incubated at 37°C for 20 minutes. 1 ml of (28%) TCA and 1.5 ml of (1%) TBA was added immediately after heating. Finally, the reaction mixture was again heated at 100°C for 15 minutes and cool at RT. After cooling, the absorbance was taken at 532nm. Percentage inhibition of lipid peroxidation (% LPI) was calculated by the following equation: % I = [(Acontrol - Asample)/ Acontrol] × 100 Where Acontrol is the absorbance of the control, and Asample is the absorbance of the extractives/standard. Then percentage of inhibition was plotted against concentration 29 2.3.5 Nitric Oxide Scavenging Activity Assay The solution of SNP (10mmol/l) in phosphate buffer solution (PBS, pH 7.4) was mixed with different concentration of extract, ascorbic acid and a-tocopherol (20_100µg/ml). The mixture was included at 37oC for 60 minutes in light. The half quantity of aliquot was taken and mixed with equal quantity of the Griess reagent and the mixture was incubated at 25oC for 30min in the dark. The absorbance of pink chromophore generated during diazotization of nitric ions with sulphanilamide and subsequent coupling with naphthyl ethylene diamine dihydrochloride was read at 546nm against a blank (Green et al., 1982). Ascorbic acid and a-tocopherol were used as standard reference compounds. The percent inhibition activity was calculated using the formula. Percent inhibition (%)= (Acontrol- Atest)/Acontrol x 100. (chio and lee, 2009). Where Acontrol is the absorbance of the control reaction at 546nm and Atest is the absorbance of a test reaction at 546nm. 2.4 Statistical Analysis In this work, all assays were done in triplicate and the data were represented as the mean ± standard deviation (SD). The statistical software Graphpad Prism Version 6.0 was applied for statistical analysis. 30 CHAPTER THREE 3.0 RESULTS 3.1. Hydrogen Peroxide (H2O2) Scavenging Activity of Moringa oleifera Leaves and Seeds Table 3.1: Showing Moringa oleifera leave and seed, Ascorbic Acid and Gallic acid Hydrogen Peroxide Scavenging Activity. Concentration (µg/ml) H2O2 scavenging % M. oleifera leave M. oleifera seed Ascorbic acid Gallic acid 100 42.86±3.19 14.29±2.83 38.09±4.01 47.62±3.61 200 57.14±5.02 19.05±3.12 57.44±3.91 52.38±5.27 400 61.90±3.74 23.81±3.70 65.66±3.68 63.27±5.42 800 71.43±4.01 33.33±3.29 76.19±4.11 80.95±8.66 1000 80.95±3.92 42.86±4.20 85.71±3.77 90.47±4.58 IC50 346.5µg/ml 501.1µg/ml 305.0µg/ml 471.9µg/ml 31 % scavenging activity 100 moringa oleifera leave moringa oleifera seed 80 ascorbic acid 60 gallic acid 40 20 0 0 500 1000 1500 concentration (µg/ml) Figure 3.1: Graphical Representation of Hydrogen Peroxide (H2O2) Scavenging Activity of Moringa oleifera Seeds and Leaves 32 3.2 Hydroxyl Radical Scavenging Activity of Moringa oleifera Leaves and Seeds Table 3.2: Showing Moringa oleifera Leave and Seed, Ascorbic Acid and Gallic Acid Hydroxyl Radical Scavenging Activity. Concentration Hydroxyl radical (µg/ml) scavenging % M. oleifera seed Ascorbic acid Gallic acid 100 17.89±2.97 34.00±2.13 34.22±3.97 33.33±0.01 200 22.15±2.05 52.34±3.02 52.57±2.36 52.57±2.36 400 28.63±3.17 69.57±2.81 69.79±3.21 43.84±2.82 800 32.43±2.94 70.46±3.02 70.69±4.83 67.33±2.79 1000 36.24±3.11 71.14±5.47 71.58±5.41 68.45±3.88 IC50 350.7µg/ml 201.4 µg/ml 202.1 µg/ml 323.0 µg/ml hydroxyl radical scavenging M. oleifera leave 100 moringa oleifera leave moringa oleifera seed 80 ascorbic acid 60 gallic acid 40 20 0 0 500 1000 1500 concentration (µg/ml) Figure 3.2: Graphical Representation of Hydroxyl Radical Scavenging Activity of Moringa oleifera Leaves and Seeds 33 3.3 Lipid Peroxidation (LPO) Inhibitory Activity of Moringa oleifera Leave sand Seed Table 3.3: Showing Moringa oleifera Leave and Seed, Ascorbic Acid and Gallic Acid Lipid Peroxidation (LPO) Inhibitory activity Concentration Lipid Peroxidation (µg/ml) (Lpo) Inhibitory activity % M. oleifera M. oleifera seed Ascorbic acid Gallic acid leave 41.16±4.37 43.65±2.94 38.12±3.80 43.09±2.26 200 45.30±3.13 48.89±5.07 44.48±3.71 48.62±3.15 400 58.29±3.08 58.01±4.23 46.96±5.82 56.35±2.82 800 71.55±4.71 66.85±4.22 50.83±4.26 70.44±4.79 1000 74.59±3.6 71.27±3.94 56.63±2.84 72.38±3.92 IC50 388.0µg/ml 378.1 µg/ml 376.1µg/ml 395.7µg/ml lipid peroxidation inhibition 100 100 moringa oleifera leave moringa oleifera seed 80 ascorbic acid 60 gallic acid 40 20 0 0 500 1000 1500 concentration (µg/ml) Figure 3.3: Graphical Representation of Lipid Peroxidation Inhibition of Moringa oleifera Leaves and Seed 34 3.4 Total Antioxidant Capacity of Moringa oleifera Leave and Seed Table 3.4: Showing Moringa oleifera Leave and Seed, Ascorbic Acid and Gallic Acid Total Antioxidant Capacity Concentration Total (µg/ml) Antioxidant Capacity % M. oleifera seed Ascorbic acid Gallic acid 100 28.94±3.54 15.78±4.32 31.57±2.94 13.15±2.26 200 31.57±2.98 42.11±3.14 36.84±5.2 31.57±5.40 400 36.84±2.18 63.15±3.80 44.73±3.06 63.15±3.41 800 55.26±1.45 60.52±3.82 55.26±3.13 65.78±3.20 1000 52.63±3.79 63.15±4.01 65.78±4.64 76.31±3.17 IC50 465.5µg/ml 198.8 µg/ml 471.7 µg/ml 271.6 µg/ml total antioxidant capapcity M. oleifera leave 100 moringa oleifera leave moringa oleifera seed 80 ascorbic acid 60 gallic acid 40 20 0 0 500 1000 1500 concentration (µg/ml) Figure 3.4: Graphical Representation of Total Antioxidant Capacity of Moringa oleifera Leaves and Seeds 35 3.5 Nitric Oxide Scavenging Activity of Moringa oleifera Leave and Seed Table 3.5: Showing Moringa oleifera Leave and Seed, Ascorbic Acid and Gallic Acid Nitric Oxide Scavenging Activity. Concentration Nitric Oxide (µg/ml) Scavenging % M. oleifera leave M. oleifera seed Ascorbic acid Gallic acid 100 19.61±4.43 21.17±4.01 25.29±3.32 27.35±2.69 200 30.78±4.61 27.25±3.34 31.56±2.75 33.03±3.02 400 32.06±5.70 30.78±5.77 38.92±2.17 41.07±2.61 800 37.15±3.80 42.94±5.83 41.47±2.38 44.70±2.14 1000 42.05±4.77 44.31±6.21 48.92±2.24 50.29±1.95 IC50 289.3µg/ml 388.6 µg/ml 363.3 µg/ml 347.9 µg/ml nitric oxide scavenging 60 moringa oleifera leave moringa oleifera seed gallic acid 40 ascorbic acid 20 0 0 500 1000 1500 concentration (µg/ml) Figure 3.5: Graphical Representation of Nitric Oxide Scavenging Activity of Moringa oleifera Leaves and seeds 36 Table 3.6: GC-MS Spectral Analysis of Crude Water Soluble Polysaccharide Extract of Moringa oleifera Leave Table 3.6 shows the crude water soluble polysaccharide extract of Moringa oleifera leaves which contains rich phytochemicals and up to 19 compounds which was identified by the Gas Chromatography-Mass Spectrometer according to their peak number, retention time, and molecular mass. Peak No. RT (min) Isolated Compounds Molecular Molecular Formular Mass (g/mol) C3H9BO3 103.913 1 2.368 Boric acid, trimethyl ester 2 2.838 1-(2-Methoxyethoxy)-2-methyl- C7H16O3 148.20 2-Propanol 3 3.407 1,2,3,4-Undecanetetrol, [2R- C11H24O4 - (2R*,3S*,4S*)]4 3.657 1-Butanol C4H10O 74.12 5 3.857 o-Ethylhydroxylamine C2H7NO 61.08 6 4.201 Cyclotrisiloxane, hexamethyl C6H18O3Si3 222.46 7 4.564 Silanediol, dimethyl C2H8O2Si 92.17 8 5.434 Silane, C10H16OSi 180.32 C21H15N 281.3 C10H30O5Si5 370.77 trimethyl(phenylmethoxy)9 6.115 7H-Dibenzo[b,g]carbazole, 7methyl 10 9.575 Cyclopentasiloxane, 37 decamethyl11 11.295 Cyclohexasiloxane, C12H36O6Si6 444.92 C14H42O7Si7 519.07 C20H40 280.313 C13H26O2 - C17H30OSi 278.5 C22H19N5O 369.4 dodecamethyl12 12.189 Cycloheptasiloxane, tetradecamethy 13 12.452 Cyclohexane, 1-(1,5dimethylhexyl) -4-(4methylpentyl)- 14 12.746 want you to know that I can be sometimes a strict mistress, but never cruel. I mean, I will treat you very well, despite the fact you will be whipped, spanked and enjoyed 15 12.996 Phenol, 2,4-bis(1,1dimethylethyl) 16 13.447 1-(4-Acetamidoanilino)-3,7dimethylbenzo[4,5]imidazo[1,2a]pyridine-4-carbonitrile 17 13.565 Methyl tetradecanoate C15H30O2 242.40 18 14.097 2-Thiopheneacetic acid, 4- C20H34O2S 338.5 tetradecyl ester 38 19 14.304 9-Octadecenoic acid (Z)-, methyl ester 39 C19H36O2 - Figure 3.6: Chromatogram of Compounds Identified in the Crude Water Soluble Polysaccharide Extracted from Moringa oleifera Leaves 40 Table 3.7: GC-MS Spectral Analysis of Crude Water Soluble Polysaccharide Extract of Moringa oleifera Seed Table 3.7 shows the crude water soluble polysaccharide extract of Moringa oleifera Seeds which contains rich phytochemicals and up to 21 compounds which was identified by the Gas Chromatography-Mass Spectrometer according to their peak number, retention time, and molecular mass. Peak No. 1 RT (min) 2.343 Isolated Molecular Molecular Compounds Formular Mass (g/mol) Thiocyanic acid, C2H3NS 73.12 methyl ester 2 2.869 Silane, dimethyl C2H6Si 58.15 3 3.407 Methyl 3-O-methyl- C6H12O5 164.16 .beta.-Dxylopyranoside 4 3.669 1-Butanol C4H10O 74.12 5 3.857 o- C2H7NO 61.08 C6H18O3Si3 222.46 C2H8O2Si 92.17 Ethylhydroxylamine 6 4.207 Cyclotrisiloxane, hexamethyl- 7 4.545 Silanediol, dimethyl 41 8 5.421 Benzyl alcohol, C10H15BrOSi 259.21 C8H24O4Si4 296.61 C6H16Pb 295 C11H22O2 - C12H36O6Si6 444.92 C18H36O2 284.5 C14H42O7Si7 519.07 C21H40O2 324.5 C13H26O2 - C14H22O - bromomethyldimethy lsilyl ether 9 6.121 Cyclotetrasiloxane, octamethyl- 10 9.574 Plumbane, diethyldimethyl 11 9.987 Decanoic acid, methyl ester 12 11.295 Cyclohexasiloxane, dodecamethyl 13 11.801 Tetradecanoic acid, 5,9,13-trimethyl-, methyl ester 14 12.189 Cycloheptasiloxane, tetradecamethyl 15 12.464 i-Propyl 11octadecenoate 16 12.740 Dodecanoic acid, methyl ester 17 12.990 Phenol, 2,5-bis(1,1dimethylethyl) 42 18 13.215 Diethyl Phthalate C12H14O4 222.24 19 13.421 Pentasiloxane, C12H36O4Si5 384.84 C15H30O2 242.40 C20H40 280.5 dodecamethyl 20 13.553 Methyl tetradecanoate 21 14.147 3-Eicosene, (E)- 43 Figure 3.7: Chromatogram of Compounds Identified in the Crude Water Soluble Polysaccharide Extracted from Moringa oleifera seeds 44 CHAPTER FOUR 4.0 DISCUSSION Free radicals have been a subject of critical interest among researchers in the previous decade. The wide range of free radical effects in biological systems has garnered interest from many specialists. It has been demonstrated that free radicals assume an important role in the pathogenesis of specific diseases and aging (Naama et al., 2013; Kadhum et al., 2011). Numerous synthetic cancer prevention agents have presented toxic and/or mutagenic effects; thus, naturally occurring antioxidants have been considered (Pal et al., 2012). Oxidative stress, “state in which oxidation exceeds the antioxidant systems in the body secondary to a loss of the balance between them.” It not only causes hazardous events such as lipid peroxidation and oxidative DNA damage, but also physiologic adaptation phenomena and regulation of intracellular signal transduction. Oxidative stress can influence many biological processes such as apoptosis, viral proliferation, and inflammatory reactions. In these processes, gene transcription factors such as nuclear factor and activator protein-1 (AP-1) act as oxidative stress sensors through their own oxidation and reduction cycling. This type of chemical modification of proteins by oxidation and reduction is called reduction-oxidation (redox) regulation (Toshikazu and Yuji, 2002). The compounds in the crude water soluble polysaccharide extracted from Moringa oleifera leaves and seeds were evaluated using GCMS and the free radical scavenging activities of the extract were evaluated using standard protocols. 19 and 21 compounds were tentatively identified in the extract using the Gas Chromatography – Mass Spectrometer by comparing the retention time, peak number and molecular mass in references or available standards 45 In this study, the free radical scavenging activity of crude water soluble polysaccharide extracted from Moringa oleifera leaves and seeds were evaluated. H2O2 is highly important because of its ability to penetrate into biological membranes. H2O2 itself is not very reactive, but it can sometimes be toxic to cell because it may give rise to hydroxyl radicals in the cells (Gulcin et al., 2010). Scavenging of H2O2 by extracts may be attributed to their phenolics, which can donate electrons to H2O2, thus neutralizing it to water (Ebrahimzadeh et al., 2009). However, the IC50 (µg/ml) of hydrogen peroxide scavenging activity of Moringa oleifera leaves is 346.5µg/ml while that of the seeds is 501.1µg/ml compared to the standards (Ascorbic acid and Gallic acid) which are 305.0µg/ml and 471.9µg/ml respectively. This is as a result of the polysaccharide and phytochemical constituents of Moringa oleifera leaves and seeds. Hydroxyl radical is the most reactive oxygen centered species and causes severe damage to adjacent biomolecule. Hydroxyl radical scavenging activity was estimated by generating the hydroxyl radicals using Ascorbic acid and Gallic acid as the standards. Therefore, the scavenging activity of the hydroxyl radical is commonly used to evaluate the ability of scavenge free radicals of substance (Taihua and Cheng, 2017). The crude water polysaccharide extract of Moringa oleifera leaves and seed shows their hydroxyl radical scavenging activity at different concentration compared to the standards (see table 3.2). The IC50 extract of Moringa oleifera leaves and seeds are 350.7µg/ml and 201.4µg/ml while that of the standards (Ascorbic acid and Gallic acid) are 202.1µg/ml and 323.0µg/ml respectively. The extracts have hydroxyl radical scavenging potentials. Lipid peroxidation is an accumulated effect of reactive oxygen species (ROS), which leads to deterioration of biological systems. It may be initiated by reactive free radicals, which subtract 46 an allylic hydrogen atom from a methylene group of polyunsaturated fatty acid side chains (Dzingiral et al., 2007). The IC50 of Moringa oleifera leaves and seeds extract are 388.0µg/ml and 378.1µg/ml while that of Ascorbic acid is 376.1µg/ml and that of the Gallic acid is 395.7µg/ml. Here, it is assumed that the antioxidant activity and reducing power capacity of the extracts was likely due to the presence of polysaccharides, which can act as free radicals scavenger by donating an electron or hydrogen. Antioxidant capacity depends on several factors, such as the rate of reaction between the sample and the reactive species and the concentration ratio between antioxidant and target. (Magalhães and coworkers, 2008) reviewed the most commonly used methods for the in vitro determination of antioxidant activity and showed that they differ from each other regarding reaction conditions and mechanisms, oxidant and target/probe species, and in the form in which results are expressed. The Moringa oleifera leaves and seeds have an IC50 of 465.5µg/ml and 198.8µg/ml and the standards (Ascorbic acid and Gallic acid) have an IC50 of 471.7µg/ml and 271.6µg/ml respectively. For the Nitric oxide scavenging percentage, Moringa oleifera leaves and seeds has an IC50 of 289.3µg/ml and 388.6µg/ml respectively while Ascorbic acid and Gallic acid has an IC 50 of 365.3µg/ml and 347.9µg/ml respectively. This implies that Moringa oleifera leaves and seeds acts in a potent manner and scavenges nitric oxide free radicals better than the standards as it has the lowest IC50. The antioxidant potential and capacity of Moringa oleifera leaves and seeds are as a result of its polysaccharide contents. These results suggest Moringa oleifera leaves and seeds as a potent therapeutic drug against cancer and other diseases related to oxidative stress and inflammation. 47 4.1 CONCLUSION In conclusion, crude water soluble polysaccharide extracted from Moringa oleifera leaves and seeds act as a potent anti-oxidative and anti-inflammatory agent in-vitro as a result of their low IC50 values for different in vitro anti-oxidative and anti-inflammatory assays as compared to Ascorbic acid and Gallic acid. In the present work it was found that Moringa oleifera leaves and seeds extracts contains antioxidant and anti-inflammatory activity. 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