Reactive Oxygen Species Ros Sources Biology Essay

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According to Cheesemen and Slater, the definition of free radicals stands for a chemical species that compose an unpaired electron in the outer (valence) shell of the molecule. This is the reason why they are highly reactive. The fact that they are highly reactive means that they have a low chemical specificity that makes them to react with most molecules in its vicinity. This includes proteins, lipids, carbohydrates and DNA.

Besides that, free radical molecules gain their stability by capturing the needed electron from the surroundings, because they don't survive in their original state for very long. Hence, free radicals attack the nearest stable molecule by "stealing" its electron. When the attacked molecule loses its electron, it becomes a free radical itself and begins a chain reaction (figure 2.1). Once the process is started, it can cascade, finally resulting in the disruption of a living cell. Free radicals are produced continuously in cells either as by-products of metabolism or deliberately as in phagocytosis. (As cited by Dr. Tamer Fouad, 2007).

Figure 2.1: free radicals attack the nearest stable molecule, "stealing" its electron. When the "attacked" molecule loses its electron, it becomes a free radical itself.

2.1.1 Reactive Oxygen Species (ROS) sources

Reactive Oxygen Species (ROS) are essential intermediates in oxidative metabolism. Nonetheless, when produced in excess, ROS can damage cells by peroxidizing lipids and disrupting structural proteins, enzymes and nucleic acids. Excess ROS are generated during a variety of cell stresses, including ischemia, exposure to ionizing and ultraviolet radiation. Reactive Oxygen Species (ROS) may contribute to inflammation and tissue damage (U-C Hipler, U.Wollina, D.Denning and B. Hipler, 2002).

According to Foyer and Noctor (2009), chloroplasts, peroxisomes, and mitochondria are major sites of ROS production in the plant cell. However, Torres and Dangl (2005) stated that large amount of ROS can also be produced at the plasma membrane during the oxidative burst associated with the defense response against invading pathogens and abiotic stress. It is a general observation that ROS production increases whenever a plant cell is under stress and there is evidence that the mitochondria contribute to that increase.

In the mitochondrial Electron Transport Chain (ETC) process, the two oxidases, cytochrome oxidase interact with O2 and transfer four electrons to reduce it to water. However, oxygen can also pick up a single electron at sites in complexes I and III (Figure 2.2). When this happens, superoxide (O2-â-) is formed. Superoxide can be transformed into H2O2 by superoxide dismutase, an enzyme present in the matrix cellular, and H2O2 can in turn, give rise to the very reactive hydroxyl radical (HOâ-) if it interacts with free metal ions such as Fe2+ or Cu+. Plant mitochondria contain several enzyme systems, all using NADPH, dedicated to removing H2O2 from the matrix, as reviewed by Moller (2001 & 2007) (as cited in Dr. Frank, 2010).

Figure 2.2: Sites of superoxide formation in the respiratory chain (Julio, 2003).

O2-â- generation has been frequently implicated in the control of normal cell growth and the promotion of malignant transformation. Relatively high concentration of ROS will lead to apoptosis or necrosis, however low concentration of O2-â- and H2O2 (Johannes, 2003)

2.1.3 Endogenous ROS

Endogenous oxygen-derived free radical species occur naturally as products of aerobic metabolism produced during normal oxidative metabolism as a result of oxidase activity, flavoprotein dehydrogenases (enzyme found in mitochondria), mitochondrial ETC, cytochrome P450 activity, as well as autoxidation of thiols and hydroquinones (involve in apoptosis) (Fereidoon, 1997).

ROS in airways may originate from endogenous and /or exogenous sources. In Martin et al., (1997) reports said that endogenous sources include respiratory bursts from activated inflammatory and immune cells, normal metabolic reactions with the ETC of the mitochondria (major source), and resident airway epithelial cells themselves. As electrons are passed along the chain from protein to protein, electrons leak from the ETC onto oxygen molecules and produce superoxide anion.

One of the examples is when neutrophils and macrophages produce ROS to destroy engulfed bacterial or fungal pathogens. In neutrophils, engulfed bacteria are compartmentalized into phagosomes which fuse with ROS and hydrolytic enzyme rich lysosomes. The consumption of oxygen during the generation of ROS is termed the 'respiratory burst'. The respiratory burst involves activation of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which produces large quantities of O2-â-. Superoxide dismutases subsequently catalyze the conversion of O2-â- anions to H2O2 (Sadis, 2008).

2.1.4 Exogenous ROS

Exogenous sources of ROS include exposure to environmental air pollutants; ozone, heavy metals (such as mercury, cadmium, and lead) and cigarette smoke (Sadis, 2008). Exposure to ionizing radiation (from industry, sun exposure, cosmic rays, and medical X-rays) can damage DNA directly, but the predominant pathway arises from radiolysis of H2O, which results in the formation of ROS that can react with DNA (Charles, 2007).

2.1.5 Effect of ROS on human Health

Dr. Denham Harman (1950's) first proposed the free radical theory of aging. He proposes that the metabolic energy production produces toxic byproducts called free radicals, which damage cellular DNA and are a primary cause of aging. When DNA is damaged, it will affect the function of cells during proliferation process and subsequently form new cells. This process can lead to cancer when the cells divide with bad DNA encoding. Aging can thus be thought of as cells replicating with bad instructions.

Free radicals have different effects on body structures such as:


Damage to the cardiovascular system

Damage to the immune system

Damage to the skin and organ through collagen cross linking

Damage to the brain and cognitive functions.

Free radical reactions are expected to produce progressive adverse changes when they accumulate throughout the body. Such changes with age are relatively common to all. However, superimposed on this common pattern are patterns influenced by genetics and environment changes that modulate free radical damage. It is possible that endogenous free radical reactions, such as ionizing radiation may result in tumour formation.

The highly significant correlation between consumption of fats and oils and death rates from leukaemia and malignant neoplasia of the breast, ovaries and rectum among old people (over 55 years old) may be a reflection of greater lipid peroxidation (studies of Lea AJ, 1966). Studies on atherosclerosis by Harman D. (1992) reveal the probability that the disease may be due to free radical reactions involving diet-derived lipids in the arterial wall that cause serum to produce peroxides and other substances. These compounds induce endothelial cell injury and produce changes in the arterial wall. (as cited in K. Bagchi & S. Puri, 1998).

2.2 Oxidative stress and lipid peroxidation

Depletion of antioxidant defenses and/or rises in ROS production can tip the ROS-antioxidant balance and cause oxidative stress, which may result in tissue injury. Oxidative stress can produce major interrelated derangements of cell metabolism, including:

DNA-strand breakage (often an early event)

Rises in intracellular "free" Ca2+

Damage to membrane ion transporters and/or other specific proteins

Peroxidation of lipids. (attack on the fatty acid)

According to Stainberg (1989), Lipid peroxidation appears to be a highly significant consequence of oxidative stress in injured human arterial walls, contributing to the development of atherosclerotic lesions. Studies done by many researches showed that damage by oxidative stress is rarely mediated by accelerating the bulk peroxidation of cell membrane lipids, for an example, the sequence of reaction is often not

Oxidative stress lipid peroxidation cell damage

But may be more usually

Oxidative stress cell damage secondary increased lipid peroxidation of damaged cells.

Lipid peroxidation is often a late event, accompanying rather than causing final cell death. However, Steinberg and Recknagel (1989) stated that lipid peroxidation does appear to make a significant contribution to the development of atherosclerosis, a major clinical problem or ischemic injury to the brain or spinal cord, aging and etc. Detection of end products of lipid peroxidation is the evidence most frequently quoted for a role of free radicals in human disease or tissue injury by toxins, and an understanding of peroxidation is of major importance in the food industry. (Barry & Susanna, 1993).

2.3 Antioxidant

Antioxidants are substances that will prevent potential diseases, producing cell damage that come from natural body processes and from exposure to certain chemicals. The body can produce its own antioxidants and also obtain them directly from food. Antioxidants are abundant in vegetables and fruits and are also found in grain cereals, teas, legumes, and nuts. Example of antioxidants includes anthocyanins, beta-carotene, cathechins, coenzyme Q10, flavonoids, lipoic acid, lutein, lycopene, selenium, and vitamin C and E. Many antioxidants are also available as dietary supplements (National Institute of Health, 2010).

The chemical nature of any antioxidant determines its solubility, and thus its localization in biological tissues. For example, lipid soluble antioxidants are localized in membranes and functions to protect against oxidative damage of membrane. Water-soluble antioxidants located in the cytosol, mitochondria matrix or extracellular fluids. They may not access to ROS generated in membranes. Vitamin C, glutathione, uric acid and lipoic acid are most commonly known water soluble antioxidants. Vitamin E and A, coenzyme Q, carotenoids, flavonoids, and polyphenols represent the most extensively studied naturally occurring fat soluble antioxidants (Chandan, Lester & Osmo, 2000).

2.3.1 Types of antioxidant

Antioxidants are believed to prevent the oxidation of fats and oils by donating hydrogen. This can be accomplishing by either natural or synthetic antioxidants. Examples of natural antioxidants are vitamin E and ascorbic acid while examples of synthetic antioxidants are butylated hydroxytoluene, butylated hydroxyanisole and ethoxyquin (John W., 1989). Besides this, antioxidant also can be classifies into two groups: endogenous antioxidant and exogenous antioxidant. Examples of endogenous antioxidants are superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (GSH-Px). Examples of exogenous antioxidants are vitamin C, vitamin E and Beta-carotene.

2.3.2 Antioxidant Nutrients

The primary function of vitamin C (ascorbic acid) is the production of collagen, which forms the basis for connective tissue in bones, teeth, and cartilage. It also plays a vital role in wound healing, immunity, and the nervous system, and acts as a water-soluble antioxidant. Because vitamin C is water-soluble, its antioxidant functions take place in aqueous body compartments. It also helps protect low-density lipoprotein cholesterol (LDL-C) against free radical damage. As an antioxidant, it helps protect against cancer and certain effects of aging. Severe deficiency of vitamin C leads to scurvy, which includes symptoms of bleeding gums, joint pain, easy bruising, dry skin, fluid retention, and depression (June H, 2000).

Studies indicate that vitamin E slow the aging process and prevent premature aging by prolonging the useful life of the cells, thus maintaining the function of the organs. Vitamin E utilized practically in tissues. The bulk of vitamin E is stored in muscles and fat tissues, but the highest concentration is found in the pituitary gland, adrenal gland, and testes. Much of vitamin E's usefulness seems to come from its role as a powerful antioxidants and anticarcinogen. Part of vitamin E's benefits may be due to its ability to protect vitamin A and C from oxidation, thus keeping them potent. In addition, vitamin E helps to increase the body's level of superoxide dismutase, an enzyme that is powerful free radical scavenger. Because of its antioxidant capabilities, vitamin E helps protect the body from mercury, lead, carbon tetrachloride, benzene, the ozone, nitrous oxide, and a variety of other carcinogens and toxins that bring about harm through their ability to act as free radicals (Sahri and Nancy, 2007)

Carotenoids is a colourful plant pigments which the body can turn into vitamin A and help prevent some forms of cancer and heart disease and act to enhance the immune response to infections. Its antioxidant behaviour protects the lining of the arteries and fats in the blood from free radical's oxidative damage and age-related macular degeneration of the eye, which leads to vision loss. Beta-carotene is used to treat skin problems caused by sun exposure such as swelling, redness, itching and pain. Typically this is the result of excessive free radical damage due to a cellular problem (Jennifer, 2010).

2.3.3 Antioxidant Enzymes

The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) serve as the primary line of defense in destroying free radicals. SOD first reduces (add an electron) the radical superoxide (O2-) to form hydrogen peroxide (H2O2) and oxygen (O2) (equation 2.1).

2O2- + 2H -SOD H2O2 + O2 Equation 2.1

Catalase and GSH-Px (equation 2.2) then work simultaneously with the protein glutathione to reduce hydrogen peroxide and ultimately produce water (H2O).

2H2O2 -CAT H2O + O2 Equation 2.2

H2O2 + 2glutathione -GPx oxidized glutathione + 2H2O

The oxidized glutathione is then reduced by another antioxidant enzyme known as glutathione reductase. Together, they repair oxidized DNA, degrade oxidized protein, and destroy oxidized lipids (fat-like substances that are constitute of cell membranes). Various other enzymes act as a secondary antioxidant defense mechanism to protect the body from further damage. While primary defense mechanism are thought to interact directly with harmful free radicals, the secondary defenses mechanisms combat processes elicited by reactive oxygen species, such as lipid peroxidation, which include enzymes that breakdown proteins and lipids and DNA repair mechanisms (Ong, 2009).

2.3.4 Polyphenol Antioxidant

Polyphenols are present in a variety of plants utilized as important components of both human and animal diets. Frankel et al., (1993) stated that, polyphenols are products of the secondary metabolism of plants. Polyphenols exhibit a wide range of biological effects as a consequence of their antioxidant properties. They inhibit LDL oxidation in vitro. Besides, polyphenols probably protect LDL oxidation in vivo with significant consequences in atherosclerosis and also protect DNA from oxidative damage with important consequences in the age-related development of some cancers. These facts are supported by Halliwell (1999). In addition, flavonoids have antithrombotic and anti-inflammatory effects.

Several types of polyphenols such as phenolic acids, hydrolysable tannins and flavonoids show anticarcinogenic and antimutagenic effects. Polyphenols might interfere in several of the steps that lead to the development of malignant tumors, inactivating carcinogens, inhibiting the expression of mutant genes and the activity of enzymes involved in the activation of procarcinogens and activating enzymatic systems involved in the detoxification of xenobiotics. Several studies have shown that in addition to their antioxidant protective effect on DNA and gene expression, polyphenols, particularly flavonoids, inhibit the initiation, promotion and progression of tumors (as cited in Ines & Federico, 2000).

2.3.5 Synthetic Antioxidant

Phenolic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxylanisole (BHA), tertiary butylated hydroxyl quinone (TBHQ) and propyl gallate (PG) are synthetic antioxidants that have been used for years as food preservatives to prolong food shelf life (Table 2.1)(Su, 2006). These synthetic substances have been shown to cause several diseases, such as enlargement of liver, reduce of food intake, growth inhibition and etc. studies performed by Lehman et al., (1995) have shown that rats and mice fed high dose levels of synthetic antioxidants produced hyperplasia of the fore-stomach and showed growth retardation (as cited in Leong & Shui, 2002).

Table 2.1: Type of synthetic antioxidants and their usages (Asim Kumar, 2007).

Synthetic antioxidant

Typical uses

L-Ascorbic acid (vitamin C)

Fruit juice, drinks mayonnaise, cured meat, fish products, butter etc.

Synthetic α-tocopherol

Infant foods, milk, fat, mayonnaise

Propyl gallate (PG)

Chewing gum, vegetables oils

Butylated hydroxy anisole (BHA)

Animal fats, cheese spread, biscuits, potato flakes, beef stock cubes.

Butylated hydroxy toluene (BHT)

Walnuts, chewing gum.

Citric acid

Vegetables oil, mayonnaise

Tertiary butyl hydroquinone (TBHQ)b

Palm oil, frying oil.

2.4 Pros and Cons of Antioxidant

Antioxidants have several benefits over our body. One of it is consuming sufficient quantities of antioxidants through the diet is very much important to maintain overall health, thus keep ourselves away from harmful diseases. Antioxidants have been linked to the prevention of all sorts of cancer since a very long time. Antioxidants are also vital in strengthening the immune system and therefore prevent our body from viral and bacterial infections.

The most popular reason why antioxidants are preferred by many people is their anti-aging benefits. Antioxidants prevent the aging process of the cells in the body and therefore contribute to improved body functions for a long time. Other than that, antioxidants help in preventing heart diseases and also minimize the oxidation process of cholesterol. Apart from being healthy for the heart, antioxidants are beneficial for the prevention of eye related disorders such as macular degeneration and glaucoma. Besides, antioxidants also involve in contribution towards the protection of central nervous system and maintaining cardiovascular health in the body (Ujwal, 2007).

Contrary to all this benefits, researchers have found that clinical trials have shown that there is no improvement sign in cancer and heart disease patients, even after consuming antioxidant supplement in the recommended dosage. In some people, symptoms have been worsened after taking supplement and lead to unusual side effects. This is cause by the endogenous made antioxidants such as glutathione or dietary antioxidants such as vitamin C that protect cancer cells during therapy , and low doses of individual dietary antioxidant may stimulate the proliferation of residual cancer cells (Kedar, 2004).

The studies performed by the researches in Kansas State show that some antioxidants are helpful for reducing the effects of poor oxygenation on the muscle. In some cases, they can rob the body of oxidants like hydrogen peroxide, and make the muscles function less efficient. Hydrogen peroxide makes blood vessels dilate to improve oxygen delivery. Antioxidants can take away natural vasodilators leading to poor muscles function (Kathleen, 2010).

2.5 Plant Profile

2.5.1 Lycopersicon esculentum

Figure 2.3: Various parts of Lycopersicon esculentum. Clockwise from the

top: Leaves, fruits and seeds.

Family : Solanaceae

Common name: Tomato

Habitat: Occasional escape from cultivation & may persist in waste areas.

Part used: Fruits and Seeds

Description: The genus Lycopersicon of the family Solanaceae is believed to have originated in the coastal strip of western South America. Today it is only cultivated. According to Heiser (1969) in his research paper, stated that tomato was introduced into Europe as early herbals in the mid-16th century. It was grown for the beauty of its fruit but was not often eat, except in Spain and Italy. The fruit was thought to be poisonous due to their bright and shiny fruit where a botanist mistakenly categorized it into a deadly nightshade family (Solanaceae). The colour of the fruit first noted in Italy was yellow. By the 18th century, the tomato began to be used as an edible food, although it was still listed among the poisonous plants (J. Benton, 1999).

Antioxidant properties: These plants are comprised of only five percent solids. These solids are cell walls, sugars, and acids. The rest of compartment is comprised of water (James, 2010). Tomatoes and tomato products are rich sources of folate, vitamin C, vitamin B, vitamin E and potassium. The most abundant phytonutrients in tomatoes are the carotenoids. Example of carotenoids is Lycopene which is the most prominent carotenoid followed by beta-carotene, gamma-carotene and phytoene as well as several minor carotenoids. The antioxidant activity of lycopene as well as several other carotenoids and their abundance in tomatoes makes these foods rich sources of antioxidant activity (Beecher, 1998).

Medicinal use: lycopene levels have been shown to be inversely proportional to cancers of the prostrate, cervix, pancreas and stomach due to its highest singlet oxygen-quenching capacity in vitro (antioxidant). Tomatoes are high in gamma-amino butyric acid (GABA), a compound that can help bring down blood pressure and the risk of heart disease. Tomato extracts contain effective antioxidants such as carotenoids will inactivate free radicals and slow down the progression of atherosclerosis. Scientist believes that tomato may aggravate gout problems and uric acid diseases. In Fiji, the fruit juice will be used to induce vomiting in children in case of food poisoning and to stop excessive bleeding from wounds by applying the fruit pulp. In Italy, it is used to cure scorpion and other insects' bites. In Philippines, the fresh fruit is used to treat oedema by pregnant women. The fresh fruit is used by Americans orally for kidney and liver problems, as a cathartic and also to keep good digestion (C.V Singh, 2008).

2.5.2 Abelmoschus esculentus


Figure 2.4: Various parts of Abelmoschus esculentus. Clockwise from the

top: Leaves with flower, fruits and seeds.

Family: Malvaceae

Common name: Lady's Finger, Okra

Habitat: It is a cultivated crop but it can be grown in garden under the right conditions for its fibrous fruits or "pods" (Phyllis, 2002).

Part used: Fruits and seeds

Description: Abelmoschus esculentus commonly known as okra or lady's finger is an important vegetable crop in the tropical and subtropical countries of the world. Originating in Africa, okra is widely eaten in India, Africa and the Middle East, as well as the American South, but little known in more northerly climates (Megan Stoddard, 2002). It produces a flower similar to the hibiscus plant except that its flowers are either white or pale yellow with a burgundy coloured heart. The plant bears numerous dark green coloured pods that feature small, round, mucilaginous white coloured seed arrange in vertical rows. The pods are harvested while immature and eaten as vegetable.

Antioxidant Properties: Nearly half of them which are soluble fibre in the form of gums and pectins help to lower serum cholesterol, reducing the risk of heart diseases. The other half is insoluble fiber, which helps to keep the intestinal tract healthy. The plant contains vitamin A and C. It is a good source of iron and calcium and also contains starch, fat, ash, thiamine and riboflavin.

Medicinal use: the superior fibre found in okra helps to stabilize blood sugar by curbing the rate at which sugar is absorbed from the intestinal tract. Okra's mucilage binds cholesterol and bile acid carrying toxins and sends it to liver for filtration. Okra helps lubricate the large intestine due to its bulk quantities by absorbing water and helps prevent and improve constipation. Okra fiber is excellent for feeding the good bacteria (probiotics). This contributes to the health of the intestinal tract. Besides, okra is used for healing ulcers and to keep joints limber. It helps to neutralize acids, being very alkaline, and provides a temporary protective coating for the digestive tract. In India, okra has been used successfully in experimental blood plasma replacements (Slyvia, 2009).

2.5.3 Moringa oleifera

Figure 2.5: Various parts of Moringa oleifera. Clockwise from the

top: Leaves with flower, fruits and seeds.

Family: Moringaceae

Common name: horseradish tree, drumstick tree, benzolive tree, kelor, marango, mlonge, moonga, mulangay, nébéday, saijhan, sajna or Ben oil tree.

Habitat: found wild through the plains especially hedges and in house yards. Plentiful in the sandy beds of rivers and streams

Part used: The pods

Description: Moringa oleifera is native to the sub-Himalayan tracts of India, Pakistan, Bangladesh and Afghanistan. It was utilized by the ancient Romans, Greeks and Egyptians; it is now widely cultivated and has become naturalized in many areas in the tropics. It is a perennial softwood tree with timber of low quality (Jed, 2005). Leaves are usually 3-pinnate and leaflets are in 6-9 pairs with an odd leaflet elliptic-ovate or obovate. Flowers are pedicelled, white and honey scented in spreading (Dr.Narain, 1999). The pods of Moringa are very popular vegetables in South India cuisine and valued for their distinctly inviting flavour (Rajangam, Azahakia, Thangaraj, Vijayakumar & Muthukrishan, 2001). It can be easily planted and grown with minimum care. The leaves have been said to cure and heal over 300 diseases. Leaves can be eaten fresh, cooked, or stored as dries powder for many months without refrigeration, and reportedly without loss of nutritional value. Moringa is especially promising as a food source in the tropics because the tree is in full leaf at the end of the dry season when other foods are typically scarve (Jed, 2005).

Antioxidant properties: The mature seed contains about 20% of edible oil, while the fresh pod contains ascorbic acid. Babatola and Lawal (2000) reported okra seed to be a potential source of high quality protein, a result of high lysine content while Al-wandari (1983) recommended moringa as a supplement to cereal-based diets (as cited in Effiong, Ogban, Ibia & Adam, 2009). Moringa is low in calories and is a good source of many nutrients including vitamin B6 and C, fiber, calcium, thiamine, riboflavin and folic acid. It is effective for the prevention of neural tube defects in developing foetuses mainly due to its high content of vitamin B6, calcium, fiber, and folic acid (Muray, 2005).

Medicinal use: The leaves, fruit, flowers and immature pods of this tree are used as a highly nutritive vegetable in many countries especially India. In the Philippines, it is known as 'mother's best friend' because of its utilization to increase women's milk production and is sometimes prescribed for anemia according to Estrella (2000). The widespread combination of diuretic along with lipid and blood pressure lowering constituents make this plant highly useful in cardiovascular disorders. Moringa fruit has been found to lower the serum cholesterol, phospholipids, LDL, and very low density lipoprotein (VLDL) cholesterol to phospholipid ratio. Moringa roots and seeds have antibacterial activity and are reported to be rich in antimicrobial agents. Makonnen et al. (1997) found Moringa leaves to be a potential source for antitumor activity. Moringa seeds are one of the best natural coagulants discovered so far by Ndabigengesere and Narasiah (1998) (as cited in Farooq, Sajid, Muhammad, & Anwarul, 2007).

2.5.4 Nelumbo nuciferaIMG_0073.JPG

Figure 2.6: Various parts of Nelumbo nucifera. Clockwise from the

left: Leaves with flower, and seeds.

Family: Nelumbonaceae

Common name: Kamal, renkon, hasu, lian, Egyptian lotus, teratai, sacred water-lily, sacred lotus, bunga telpok, ngau, baino, nelun ala, bua-luang.

Habitat: the red variety flower is commonly found in tanks and ponds. The white flower variety is very rare (Ajay, 2000).

Part used: the seeds

Description: Nelumbo comes from the name of the plant in Singhalese (Sri Lanka), and nucifera means "nut bearing". This water-lily is native to Asia from Iran to China and Japan, and to north-eastern Australia. The elegent, sweet-scented lotus flowers have long been regarded with reverence by Buddhists in China, India and elsewhere in the Orient. It is an aquatic, perennial herb, the lotus produces large umbrella-shaped leaves, which grow on long stalks well above the surface of the water. The flowers may be in white, pink or red. The seed bearing structure is a flat-topped receptacle in which many one-seeded carpels are embedded. The seeds are eaten raw, like nuts before they are fully ripe. When mature, the nutritious seeds, rich in vitamin C are generally roasted or boiled and may also be grounded into flour, or dried for storage. Roasted and ground lotus nuts make a tolerable substitute for coffee (Frederic, 2004).

Antioxidant Properties: The rhizome has many alkaloids. Leaves contain alkaloid nuciferin, romerin and nerenyuferin. The dried seeds contain protein 17.2%, fat 2.4% and carbohydrates 66.6%. Besides this, it also contains calcium, phosphorus, iron, ascorbic acid and sugar. The stem contains moisture 83.80%, protein 2.7%, fats 0.11%, starch 9.25%, sucrose 0.41% and calcium. Other than this, vitamin B, Vitamin C, and aspirin 2% are also present in it (Ayushveda, 2010).

Medicinal Use: Nelumbo nucifera are classified as astringents, being sweet and neutral, and benefiting the spleen, kidney, and heart. The sweet taste and nourishing qualities help to stop diarrhoea associated with qi deficiency. The astringent quality helps prevent loss of kidney essence, so the seed are used to treat weak sexual function in men and leucorrhoea in women. The seed also has calming properties that alleviate restlessness, palpitations, and insomnia. The heart of the nelumbo nucifera (embryo), is classified as bitter and cold and benefiting the heart; it dispels pathogenic heat from the heart to treat fidgets and spontaneous bleeding due to heat. The bitter components are isoquinoline alkaloids with sedative and antispasmodic effects. The alkaloids dilate blood vessels and thereby reduce blood pressure. (Subhuti, 2001).

2.5.5 Brassica oleraceaIMG_0063.JPG

Figure 2.7: Various parts of Brassica oleracea. Clockwise from the

top: Leaves, flower, and seeds.

Family: Brassicaceae

Common name: Broccoli, cauliflower, collard greens and Brussels sprouts.

Habitat: Grow wild in hills and gardens ( National Institute of Industrial Re, 2000).

Part used: Flower

Description: Brassica oleracea (broccoli) is a fast growing and branched plant. It 24-35 inches tall that produce dense green clusters of flower buds at the ends of the central axis and the branches. In Britain, the term broccoli refers to the cauliflower (another variety of Brassica oleracea). Native to the eastern Mediterranean and Asia, sprouting broccoli was cultivated in Italy in ancient Roman times and was introduced into England and America probably during colonial times. (Encyclopedia Britannica, 2011). They can be categorised into three main kinds that is smooth green, red, and savoy. The smooth green cabbages may have round, flattened or conical heads, and can be green, blue-green, or yellow-green. The red cabbages are reddish-purple and have very tight heads. The savoy cabbages have crinkled, pucker bluish-green leaves and looser heads (chinese cabbage) (Steve, 2003).

Antioxidant property: Brassica oleracea contains high levels of antioxidants and vitamins A, B1, B2, and C. it should not be over cooked because the heat will destroy much of the flavour and most of the nutrients. (Steve, 2003). Brassica provides many flavonoids in significant amount including flavonoids kaempferol and quercitin. A very high concentrated in Brassica are the carotenoids lutein, zeaxanthin, and beta-carotene. Other antioxidants provided by Brassica in beneficial amounts include vitamin E and the minerals manganese and zinc. Brassica is also concentrated in phytonutrients. One particular phytonutrient is glucosinolates is simply outstanding. The isothiocyanates (ITCs) made from Brassica's glucosinolates are the key to Brassica's cancer-preventive benefits (George, 2009).

Medicinal uses: Brassica antioxidant properties provide cardiovascular protection and contain many anti-cancer and anti-helicobacter compounds. The richness in vitamin C promotes body ability to absorb iron and calcium thus provides protection against seasonal flues. It is also rich with fibre that reduces the LDL cholesterol. The natural combination of calcium and vitamin C help in osteoporosis. Brassica detoxifies the body and removes all the toxins that cause arthritis, skin problems and allergies. Natural source of iron promotes in prevention of anemia. ( Janni, 2009).

2.7 Antioxidant Assay

There are varieties of methods to verify the antioxidant capacity. These methods differ in terms of their assay principle and experimental condition (Paixão, Perestrelo, Marques & Câmara, 2007). There are two mechanisms whereby antioxidants neutralise radicals: single electron transfer (SET) reaction and hydrogen atom transfer (HAT) reaction. The former is monitored through colour change which occurs as the oxidant is reduced whereas the latter involves the competition between the antioxidant and the substrate (probe) for free radicals (Huand, Ou & Prior, 2005).

There are methods which utilises both SET and HAT mechanisms. DPPH assay are normally categorised as SET, but can be deactivated either by direct reduction through electron transfer or by radical quenching through HAT (Jimenez, Selga, Torres & Julia, 2004). Whereas both FTC and TBA are categorised into HAT. All antioxidant assays differ from each other in terms of substrate, probes, reaction conditions, and quantization methods. It is extremely difficult to compare the results from different assay as Frankel and co-workers have already concluded (Frankel & Meyer, 2000).

2.7.1 Rapid Evaluation using Thin Layer Chromatography (TLC)

The antioxidant constituents were analysed by thin layer chromatography (TLC) using aluminium-backed TLC plates (Silica gel) followed by DPPH techniques according to Moore et al. (2006) with slightly modifications (R.Ragupathi et al., 2010). To detect antioxidant compounds extraction in various solvents, TLC plates will be sprayed with DPPH in methanol, as an indicator. The presence of antioxidant compound will be detected by yellow spots against a purple background (Miroslav, Zuzana & Janka, 2009). Various separated spots will be note as their retardation factor (Rf) values.

2.7.2 DPPH free radical Scavenging Assay

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) is a free radical with one electron delocalised over the molecule (Figure 2.3) which gives it a deep purple colour with maximum absorption of 517 nm in methanol or ethanol solution which is proportional to concentration of free radical scavenger added to DPPH reagent solution. Upon reduction, the initial dark purple solution will fade and give rise to light yellow colour due to the transfer of hydrogen atom. The decline in absorption is measured spectrophotometrically and compared with a methanol control to determine the DPPH radical scavenging activity (Shetty, 1982). DPPH can only be dissolved in organic solvent; thus, can only be applied in determining the antioxidant activity of lipophilic compounds (Wojdylo, Oszmiański & Czemerys, 2007).

Figure 2.3: Chemical Structure of DPPH radical.

2.7.3 Ferric Thiocyanate (FTC) Assay

The Ferric thiocyanate (FTC) method was used to measure the amount of peroxide at the beginning of lipid peroxidation, in which peroxide will react with ferrous chloride and form ferric ions. Ferric ions will then unite with ammonium thiocyanate and yield ferric thiocyanate. The substance is red, and dense colour is indicative of higher absorbance (Farrukh, Iqbal & Zafar, 2006).

Lipid peroxidation is produced by ROS which abstract a hydrogen atom from a methylene group of an unsaturated fatty acid and subsequently form free radicals such as peroxyl radical. Once these free radicals are formed, lipid peroxidation progresses and consequently, lipids produce various secondary products such as ketones and aldehydes ( Joon & Takayuki, 2009)

2.7.4 Thiobarbituric Acid (TBA) Assay

Among lipid peroxidation products used for antioxidant assays, malonaldehyde (MA) has been most widely used to evaluate the antioxidant activity of chemical(s) in lipid peroxidation systems. Particularly, MA is very useful as a biomarker to investigate the final stage of lipid peroxidation according to Neff et al. (1984) and Pryor et al. (1976). However, it is extremely difficult to analyse MA in a lipid sample because it is very soluble in water and tends to present as a polymer in an aqueous solution. Consequently, the MA-TBA assay became one of the most popular assays for studies related to lipid peroxidation and it is currently used widely to evaluate antioxidant activities of various natural products (Joon & Takayuki, 2009).