Antioxidant Activity Evaluation Of Methanolic Extracts

Antioxidant Activity Evaluation of Methanolic Extracts of Brassica napus by Different Assays.

A thesis submitted as partial fulfillment of the requirement for the

Degree of M.Sc. in

Chemistry

By

MUHAMMAD SHOAIB

2006-2008

DEPARTMENT OF CHEMISTRY UNIVERSITY OF SARGODHA SARGODHA, PAKISTAN

Antioxidant Activity Evaluation of Methanolic Extracts of Brassica napus by Different Assays.

A THESIS SUBMITTED TO THE

UNIVERSITY OF SARGODHA IN PARTIAL

FULFILLMENT OF THE REQUIREMENT

FOR

THE DEGREE OF M.Sc.

IN

CHEMISTRY

SUBMITTED BY

MUHAMMAD SHOAIB Roll No. 03 SESSION 2006-2008

DEPARTMENT OF CHEMISTRY UNIVERSITY OF SARGODHA SARGODHA, PAKISTAN

The Fruit of My Work is Dedicated To my Parents Whose Abundant Affections Patronizing Encouragement And Secret Prayers Have Enabled Me to Accomplish This Task And

To my Brother, Sister, friends Whose Hands Always Pray For My Success

APPROVAL CERTIFICATE

This thesis entitled “Antioxidant Activity Evaluation of Methanolic Extracts of Brassica napus by Different Assays” submitted by Mr. Muhammad Shoaib in Partial fulfillment of the requirement for the degree of Master of Science in Chemistry is hereby approved.

SUPERVISOR Dr. Rana Shahid Iqbal Asst. Professor (Analytical Chemistry) Department of Chemistry University of Sargodha Sargodha

Dr. Ilays Tariq Chairman Department of Chemistry University of Sargodha Sargodha

ACKNOWLEDGEMENT

Glory is to that Almighty Allah who has, out of a drop of fluid, created such a variety of creatures, rational and irrational! Adored be that Creator, who has established such a variety of forms, statures and vocal sounds among them, though their origin is the same pure liquid and genuine spirit. In praise of the Prophet

Muhammad, a thousand salutations and

benedictions to his sublime Holiness Muhammad Mustafa, the chosen, the benefactor – the blessing and peace of God be with Him – through whose grace the sacred Quran descended from the most high! How inadequate is man justly to praise and eulogize Him! Salutations and blessing also to His companions and posterity! I feel great pleasure in expressing my deep sense of gratitude to Dean of Sciences and Technology, Dr. Ghulam Hussain Bhatti and our respected and chairman, Department of Chemistry, University of Sargodha, Sargodha, for their constant inspiring leadership and encouragement. I, with deep emotions of benevolence and gratitude, am highly thankful to my worthy supervisor Dr. Rana Shahid Iqbal, Department of Chemistry, University of Sargodha, Sargodha, under whose dynamic supervision, illustrative advice, keen interest and sympathetic behavior, the present study was accomplished. I am grateful for her cordial behavior towards me.

I feel pleasure to express thanks to all teachers of department for being a source of inspiration for me during my research work. I offer my sincerest thanks to my affectionate parents, especially my mother, who always remembered me in her prayers and raised her hands for me to achieve the highest goal of life. This work was not possible without their moral and financial support. Words are inadequate to express my deep sense of gratitude and indebtedness to my dear class fellows, who passed two years education period of M.Sc. excellently and my best friend Muhammad Umer Farooq whose hands always raised for my success. They will remain vibrating for years in my mind for their unselfish behavior and nice company. Thanks to all non-teaching staff of Department of Chemistry, University of Sargodha, Sargodha.

Muhammad Shoaib

Sr. No.

TOPIC

Page No.

CHAPTER NO. 01

INTRODUCTION

1.1

Foods

1

1.2

Components of Foods

1

1.2.1

Proteins

1

1.2.2

Carbohydrates

1

1.2.3

Lipids

2

1.3

Fats and Oils

2

1.4

Importance for Living Organisms

2

1.5

Problems with Oils

2

1.6

Lipid Peroxidation

1.6.1

Mechanism of Peroxidation

3

1.6.1

Initiation

3

1.6.1.2

Propagation

4

1.6.1.3

Termination

5

1.6.2

Photo-oxidation

5

1.6.3

Enzymatic Peroxidation

1.7

Antioxidants

1.7.1

Desirable Qualities of Food Antioxidants

6

1.7.2

Mechanism of antioxidative action

6

1.8

Natural Antioxidants

7

3

5 6

1.8.1

Vitamin C

7

1.8.2

Vitamin E

8

1.8.3

Coenzyme Q10

9

1.8.4

Seasamol

9

1.8.5

Gossypol

10

1.8.6

Lecithin

10

1.9

Synthetic Antioxidants

10

1.9.1

Butylated Hydroxyanisole

11

1.9.2

Butylated Hydroxytoluene

11

1.9.3

Nordihydroguaiaretic Acid

11

1.9.4

Propyl Gallate

12

1.10

Superiority of Natural Antioxidants Over Synthetic

12

1.11

Sources of Natural Antioxidants

12

1.12

Brassica

13

1.12.1

Brassica napus

13

1.13

Literature Review

13

1.14

Aims and Objectives of Work

23

1.15 CHAPTER NO. 2

23

Scope of Work/Study

EXPERIMENTAL

2.1

Samples

24

2.2

Chemicals and Reagents

24

2.3

Drying and Grinding

24

2.4

Extraction of Total Antioxidants

2.6

DPPH• Scavenging Assay

24 25

2.7

Determination of Total Phenolic Contents (TPC)

2.8

Chelating Activity

2.9

Antioxidants Activity Determination in

25

25

Linoleic Acid System

CHAPTER NO. 3

25

RESULTS AND DISCUSSIONS

3. I

DPPH Radical Scavenging Abilities

26

3.2

Total Phenolic Content (TPC)

26

3.3

Chelating Activity

27

3.4

Antioxidant Activity in Linoleic Acid System

27

3.5

CHAPTER 4

Total Flavonoid Contents (TFC)

REFRENCES

27 28

CHAPTER 1 INTRODUCTION

1.1 Food Food is any substance, usually composed primarily of carbohydrates, fats, water and proteins, that can be eaten or drunk by an animal or human for nutrition. Items considered as food may be sourced from plants, animals or other categories such as fungus or fermented products like alcohol [1]. There are around 2,000 plant species, which are cultivated for food [2]. Seeds of

plants may be a good source of food for animals, including humans because they contain nutrients necessary for the plant's initial growth. In fact, the majority of food consumed by human beings is seed-based foods. Edible seeds include cereals (such as maize, wheat, and rice), legumes (such as beans, peas, and lentils), and nuts. Oilseeds are often pressed to produce rich oils, such as sunflower, rape (including canola oil), and sesame [3]. Some fruits, such as tomatoes, pumpkins and eggplants, are eaten as vegetables [4].

1.2 Components of Food Food mainly consists of proteins, carbohydrates, fats.

1.2.1 Proteins The word “protein” comes from the Greek word πρώτα ("prota"), meaning "of primary importance." Proteins are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues [6]. The end of the protein with a free carboxyl group is known as the Cterminus or carboxy terminus, whereas the end with a free amino group is known as the Nterminus or amino terminus. Protiens have primary, secondary, tertiary and quaternary structure [7]. Many proteins catalyze biochemical reactions. Proteins also have structural or mechanical functions, such as actin and myosin. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. Proteins are also necessary in animals' diets, since animals cannot synthesize all the amino acids they need [8].

1.2.2 Carbohydrate Carbohydrates (from 'hydrates of carbon') or saccharides (Greek σάκχαρον meaning "sugar") simple organic compounds that are aldehydes or ketones with many hydroxyl groups added [9]. The basic carbohydrate units are called monosaccharides, such as glucose, galactose, and fructose. If the carbonyl group is an aldehyde, the monosaccharide is an aldose; if the carbonyl group is a ketone, the monosaccharide is a ketose. Monosaccharides with three carbon atoms are called trioses, those with four are called tetroses, five are called pentoses, six are hexoses, and so on. They fill numerous roles in living things, such as the storage and transport of energy (starch, glycogen) and structural components (cellulose in plants, chitin in animals) [10]. Additionally, carbohydrates and their derivatives play major roles in the working process of the immune system,

fertilization,

pathogenesis,

blood

clotting,

and

development.

Two

joined

monosaccharides are called disaccharides (sucrose and lactose). Oligosaccharides and

polysaccharides are composed of longer chains of monosaccharide units bound together by glycosidic bonds [11].

1.2.3 Lipids Lipids are broadly defined as any fat-soluble (lipophilic), naturally-occurring molecule, such as fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, acting as structural components of cell membranes, and participating as important signaling molecules [12].

1.3 Fats and Oils Fats consist of a wide group of compounds that are generally soluble in organic solvents and largely insoluble in water. Chemically, fats are generally triesters of glycerol and fatty acids. Fats may be solid or semi-sold at normal room temperature, depending on their chemical and composition. Although the words "oils", "fats", and "lipids" are all used to refer to fats, "oils" is usually used to refer to fats that are liquids at room temperature, while "fats" is usually used to refer to fats that are solids at normal room temperature. "Lipids" is used to refer to both liquid and solid fats. Examples of animal fats are lard (pig fat), fish oil, and butter or ghee. They are obtained from fats in the milk, meat and under the skin of the animal. Examples of edible plant fats are peanut, soya bean, sunflower, sesame, coconut, olive, and other vegetable oils. These examples of fats can be categorized into saturated fats and unsaturated fats [13].

1.4 Importance for Living Organisms Fats are also sources of essential fatty acids, an important dietary requirement. Vitamins A, D, E, and K are fat-soluble compounds, meaning they can only be digested, absorbed, and transported in conjunction with fats. Fats play a vital role in maintaining healthy skin and hair, insulating body organs against shock, maintaining body temperature, and promoting healthy cell function. They also serve as energy reservoirs for the body. Fats are broken down in the body to release glycerol and free fatty acids. The glycerol can be converted to glucose by the liver and thus used as a source of energy. They yield a lot of food energy (37MJ /g, or 9 cals/g), roughly twice as much as carbohydrates [14].

1.5 Problems with Oils

On prolonged storage oils, fats and their products undergo oxidation and deterioration which results in off-odor and off-flavor. Oxidative deterioration in oils has been a problem for common interest for scientist since long. According to Hilfer, light is perhaps the most important single factor affecting the stability of oil and fats. Heat and moisture may serve as catalysts for oxidative deterioration [15].

1.6 Lipid Peroxidation Lipid peroxidation is the process whereby free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain reaction mechanism [16]. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which lie methylene -CH2- groups that possess especially reactive hydrogens. As with any radical reaction the reaction consists of three major steps: initiation, propagation and termination [17].

1.6.1 Mechanism of Peroxidation Three different mechanisms are able to induce lipid peroxidation: 1 - Autoxidation This is a radical-chain process involving three sequences.

1.6.1.1 Initiation In a peroxide-free lipid system, the initiation of a peroxidation sequence refers to the attack of a ROS (reactive oxygen species) able to abstract a hydrogen atom from a methylene group (CH2-), this hydrogen having very high mobility. This attack generates easily free radicals from polyunsaturated fatty acids. .OH is the most efficient ROS to do that attack, whereas O 2. - is insufficiently reactive.

This peroxidation process is inhibited by tocopherols, mannitol and formate. The presence of a double bond in the fatty acid weakens the C-H bonds on the carbon atom adjacent to the double bond and so makes H removal easier.The carbon radical tends to be stabilized by a molecular rearrangement to form a conjugated diene.

Under aerobic conditions conjugated dienes are able to combine with O 2 to give a peroxyl (or peroxy) radical, ROO

.

1.6.1.2 Propagation

As a peroxyl radical is able to abstract H from another lipid molecule (adjacent fatty acid), especially in the presence of metals such as copper or iron, thus causing an autocatalytic chain reaction. The peroxyl radical combines with H to give a lipid hydroperoxide (or peroxide). This reaction characterizes the propagation stage.

Probable alternative fates of peroxyl radicals are to be transformed into cyclic peroxides or even cyclic endoperoxides (from polyunsaturated fatty acids such as arachidonic or eicosapentaenoic acids)

1.6.1.3Termination Termination (formation of a hydroperoxide) is most often achieved by reaction of a peroxyl radical with a-tocopherol which is the main lipophilic "chain-breaking molecule" in the cell membranes. Furthermore, any kind of alkyl radicals (lipid free radicals) L . can react with a lipid peroxide LOO. to give non-initiating and non-propagating species such as the relatively stable dimers LOOL or two peroxide molecules combining to form hydroxylated derivatives (LOH). Some bonds between lipid peroxides and membrane proteins are also possible.

1.6.2 Photo-oxidation As singlet oxygen (1O2) is highly electrophilic, it can react rapidly with unsaturated lipids but by a different mechanism than free radical autoxidation. In the presence of sensitizers (chlorophyll, porphyrins, myoglobin, riboflavin, bilirubin, erythrosine, rose bengal, methylene blue...), a double bond interacts with singlet oxygen produced from O2 by light. Oxygen is added at either end carbon of a double bond which takes the trans configuration. Thus, one possible reaction of singlet O2 with a double bond between C12 and C13 of one fatty acid is to produce 12- and 13hydroperoxides. The lifetime of singlet O2 in the hydrophobic cell membrane is greater than in aqueous solution. Furthermore, photo-oxidation is a quicker reaction than autoxidation since it was demonstrated that photo-oxidation of oleic acid can be 30 000 times quicker than autoxidation and for polyenes photo-oxidation can be 1000-1500 times quicker. Similar effects have been described in liposomes and in intact membranes. The inhibition of photosensitized

oxidation is efficiently inhibited by carotenoids, the main protective role played by these compounds in green plants. The inhibitory mechanism is thought to be In contrast, tocopherols inhibit this oxidation by quenching the previously formed singlet oxygen, this forms stable addition products. Unexpectedly, It was shown that carotenes are efficient inhibitors in vegetal oils only if TOCOPHEROLS are also present to protect the former [18].

1.6.3 Enzymatic Peroxidation Lipoxygenase enzymes (from plants or animals) catalyze reactions between O2 and polyunsaturated fatty acids, such as arachidonic acid (20:4 n-6), containing methylene interrupted double bonds. When 20:4 n-6 is the substrate, these hydroperoxides are known as HpETEs which can be transformed into hydroxy products (HETES). These HETEs are also formed directly via cytochrome P450 induced reactions (monooxygenases) and sometimes also via cyclooxygenase enzymes.Six hydroperoxides (5-, 8-, 9-, 11-, 12-, and 15-HpETE) are known to be formed from arachidonic acid in animal cells. Dihydroperoxy compounds (DiHpETEs) may also be formed via the action of 5- and 15lipoxygenases. These compounds are important metabolic intermediates but are also bioactive.Cyclooxygenase enzymes (in plants and animals) catalyze the addition of molecular oxygen to various polyunsaturated fatty acids, they are thus converted into biologically active molecules called endoperoxides (PGG, PGH), intermediates in the transformation of fatty acids to prostaglandins.

Among the cytochrome P450 catalyzed reactions, the fatty acid epoxygenase activity produces epoxide derivatives. Those formed from 20:4 n-6 (5,6-, 8,9-, 11,12-, 14,15-EpETrE) have been shown to have prominent biological activities. Furthermore, these mono-epoxides are susceptible to be metabolized into di-epoxides, epoxy-alcohols or oxygenated prostaglandins [19].

1.7 Antioxidants An antioxidant is a substance capable of slowing or preventing the oxidation of other molecules. Antioxidants are used to preserve the edible oils and fats. An antioxidant gives hydrogen to the free radical. When the free radicals take the hydrogen atoms from antioxidant the chain is broken and reaction is stopped. In this way antioxidants are useful for the preservation of edible oils [20].

1.7.1 Desirable Qualities of Food Antioxidants An ideal antioxidant should satisfy the following requirements. It should be active in very low concentration i.e.01-.001% The used compound should be non-toxic and so for oxidation products. It should be easily incorporated into the substrate. It should impart no foreign flavor, odor and color to food even after prolonged heating and storage. Its antioxidant activity should not be limited to the fats or oils in which it is incorporated, but should be transmitted to the foods and subsequently might be prepared from this fat. It should be easily available and cost so little that its use should not significantly increase the price of food. To control its use in food, the antioxidant should be easy to detect, identify and measure [21].

1.7.2 Mechanism of Antioxidative Action Antioxidation can be represented by a chain reaction as given below.

.

R-H_________________R +H

.

.

R +O=O____________ROO

.

.

ROO +R-H_________ROOH+R

.

R +R

.______________

R-R

.

Hence there are two ways in which this chain reaction reaction can be initiated, on the one hand, addition of reagents which retard the formation of free radicals, and on the other hand the addition of free radicals accepters called antioxidants. The general principles of chain termination by free radical accepters in autoxidation reactions have been clearly recognized by Backstrom. The first detailed kinetic study was carried out by Balland.their system consisted of autoxidizing ethyllinoleate containing benzoyl peroxide and ethyl lioleate hydroperoxide as an initiator and hydroquinone as an inhibitor.

1.8 Natural Antioxidant They work together to provide the ultimate protection. When a natural antioxidant grabs a free radical, it becomes a weak free radical itself, and another antioxidant will help regenerate it. There are 5 basic natural antioxidants: Vitamin C Vitamin E Coenzyme Q 10 Lipoic acid Glutathione

1.8.1 Vitamin C Essential for the production of collagen, the cellular glue that keeps cells attached together. It strengthens the connective tissue. Thought to protect against cataract, against lipid oxidation Essential for immune system health. Has the important job of recharging fat-soluble vitamin E when it becomes a free radical itself [22].

The pharmacophore of vitamin C is the ascorbate ion. In living organisms, ascorbate is an antioxidant, since it protects the body against oxidative stress, and is a cofactor in several vital enzymatic reactions [23].

1.8.2 Vitamin E Vitamin E is a family of molecules composed of four different tocopherols and four different tocotrienols, all nearly identical in structure. Particularly high levels of vitamin E can be found in the following foods [24]. Wheat germ, Red Palm Oil, Corn, Nuts, Seeds, Olives Spinach and other green leafy vegetables.

CH3 HO CH3 H3C

CH3

CH3

O CH3

CH3

CH3

Most vitamin supplements contain only one kind of Vitamin E-alpha-tocopherol but not the others [25]. Many fats and oils are quite stable to oxidative rancidity. Oils containing antioxidants when mixed with other fats, tends to protect the fats from oxidation. For example, the tocopherol (α, beta, gamma and delta) appear to be principal antioxidants in a number of vegetable oils. They are effective stabilizer for animal fats but have little antioxigenic effect when added to vegetable fats. The alpha isomer has the greatest antioxidant activity than others [26]. Alpha tocopherol

CH3 HO

CH3

H3C

O CH3

CH3

CH3 CH3

CH3

Beta tocopherol

CH3 HO

CH3 O

CH3

CH3 CH3

CH3

CH3

Gamma tocopherol

HO

CH3 O

H3C

CH3

CH3

CH3

CH3

CH3

Delta tocopherol

HO

CH3 O CH3

CH3

CH3

CH3 CH3

1.8.3 Coenzyme Q10 This vitamin-like substance is, by nature, present in most human cells except red blood cells and eye lens cells. Ninety-five percent of all the human body’s energy requirements (ATP) are converted with the aid of CoQ10. CoQ10

Because of its ability to transfer electrons and therefore act as an antioxidant, Coenzyme Q is also used as a dietary supplement [27].

1.8.4 Seasamol Seasamol seed oil exhibits antioxidant property when added to other fats. The active antioxidant of oils is seasamol which is present in unsaponifiable matter of sesamol seed oil [28].

O O

OH

1.8.5 Gossypol Crude cotton oil contains the natural antioxidant gossypol. However, the toxic property of gossypol prevents its use as an antioxidant in edible oils and fats [29].

O HO

O

OH

OH HO HO

OH

H3C

CH3 CH3

CH3

H3C

1.8.6 Lecithin Lecithin is one of the first antioxidant to receive serious consideration in the united state for use in edible oils. Commercial lecithin preparation have been found to be somewhat effective in vegetables oils like cotton seed oil but are relatively ineffective

CH2

CH2 CH3 H3C

+

N

CH3

O

OH CH2

CH2

O P+

O

HO

CH2

C

CH2

CH2

CH2

CH2

CH

CH2

CH2

CH2

CH2

CH

in lard [30]

CH2

CH3

CH2

CH2

CH OH CH2HC CH2 O C O

1.9 Synthetic Antioxidants

CH2

CH2

CH2

CH2

CH2

CH2

CH CH

CH2 CH2

CH2 CH2

CH2

CH2 CH3

Most of the antioxidants occurring naturally in food stuff exhibit comparatively weak antioxigynic properties. Consequently a number of substances possessing marked antioxigenic properties have been developed and put in the market for use in food. Different antioxidants vary in their effectiveness to stabilize fats or fats products are used which are discussed as under.

1.9.1 Butylated Hydroxyanisole (BHT) This commercial product is a mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4methoxyphenol. These compounds do not occur naturally but can readily synthesize by butylation of para methoxy phenol.

OCH3H3C

OCH3

CH3

CH3

OH H3C

CH3

CH3

OH

They are very soluble in fats and oils but practically insoluble in water. The antioxidant property of 3-BHA is greater than 2-BHA. The most important property of BHA which accounts for its great popularity as a food antioxidant is its ability to remain effective in baked and in fried foods. It is used in low concentration due to its phenolic smell [31].

1.9.2 Butylated Hydroxy Toluene (BHT) Butylated hydroxyl toluene, commonly known as BHT is 2,6-di-tert-butyl-4-methyl phenol or 2,6-di-tert-butyl-p-cresol. It is also synthetic antioxidant, originally developed for use in petroleum products and rubber, which has been adopted for use in food products. Like BHA, BHT belongs to group of compounds called “hindered phenols” [32]

H3C

CH3 OH H3C

CH3

H3C

CH3

CH3

1.9.3 Nordihydroguaiaretic Acid This acid commonly known as NDGA was isolated in 1942 from a desert plant larrea divaricata. Pure NDGA is white crystalline solid melts at 184-1850C and very slightly soluble in water and dilute acid NDGA is effective in preventing oxidative rancidity in fat aqueous system [33].

1.9.4 Propyl Gallate It is an antioxidant approved by the meat inspection division, US department of agriculture use as edible oil in concentration .01%.. Propyl gallate is the one of the most widely used antioxidant at present and is a component of many commercial antioxidant preparations. OH

O

HO O HO

CH3

1.10 Superiority of Natural Antioxidants Over Synthetic The oils with higher content of unsaturated fatty acids, especially polyunsaturated FA, are most susceptible to oxidation. In order to overcome the stability problems of oils and fats synthetic antioxidants, such as butylate hydroxyanisole (BHT), butylated hydroxy tolune (BHT), tertiary butyl hydroquinone (TBHQ) have been used as food additives. But resent reports reveal that these compounds may be implicated by health risks, including canceer and carcinogenesis [34]. Therefore the most poweful synthetic antioxidant (TBHQ) is not allowed for food application in Japan, Canada and Europe. Similarly, BHA has also been removed from the generally recognised as safe (GRAS) list of compounds [35]. Due to these safty concerns, there is an increasing trend among food scientists to replace these synthetic antioxidants with natural ones, which in general are supposed to be safer.

1.11 Sources of Natural Antioxidants The effectiveness of different natural sources in stablizing vegetable oils has been previously studied [36]. Jung, Lee, Hun, Kyung and Chung (2001) evaluated the effect of natural lecithin on the stability of borage oil. Shahidi and Wanasundara (1992) investigated the stabilization of canola oil with canola meal. Fruits, vegetables, nuts, seeds, and bark are being investigated for their antioxidatnt potential (Pratt and Hudson, 1990). Peschel, W.et al (2007) searched natural antioxidants from vegetable and fruit wastes. Eleven fruit and vegetable byproducts and two minor crops were screened for antioxidant activity. .

Esposito, F.et al (2007) worked on

antioxidant activity and dietary fiber in durum wheat bran by-products. . Abdalla, A.E.M. et al (2006) collected Egyptian mango seeds as wastes from local fruit processing units and checked their antioxidant potential. Anna.P.et al (2009) evaluated of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Maier, T.et al (2009) observed antioxidant potential of seven grape seed samples originating from mechanical seed oil extraction. Besides these, search of newer sources of natural antioxidants from economical materials, agricultural wastes is hot area of research in recent years. As a step towards series of investigations in the said dimension, antioxidant potential of Brassica napus has been studied in this project.

1.12 Brassica Kingdom

planate,

Division

magnolipophyta

Class

magnolyopsida

Order

capparales,

Family

brasicacae.

The four important species of brassica are namely brassica napus, brassica juncea, brassica oleraceae and brassica compestriss. Brassica has 350 genera and 2500 species.

1.12.1 Brassica Napus It is also known as rapeseed, rapa (Latin for turnip .i.e. rapum, or rapa) and canola from Can-O-L-A (Canadian oil seed low acid). Leading producer include Canada, USA, Australia, China, India and Pakistan. This plant has flat leaves 12-20 inches long and 8-25 inches wide all stand 2-4 feet tall at most and yellow flowers with 4-petals [37]. Pharmaceutically leaves are used as potherb. Oil of napus seed is used in the production of erucic acid which is in turn used in the manufacture of other chemicals. Seed powdered with salt is used to be a folk remedy for cancer [38]. .Rape oil is used in massage and oil baths which is beleaved to be strengthen the skin and keep it cool and healthy. With camphor it is applied for rheumatism and stiff joints. Roots are used for chromic coughs and bronchial catarrhs. Roots are used as emollient and diacritic. Canola oil is rich with omega-6 and omega-3 fatty acids in the ratio of 2:1and has been reported to reduce cholesterol level lower serum, triglyceride level and keep platelets by sticking together [39].Per 100 g, the leaf is reported to contain 61 calories, 83.3 g H2O, 2.9 g protein, 1.7 g fat, 11.2 g total carbohydrate, 1.8 g fiber, 0.9 g ash, 136 mg Ca, 38 mg P, 4.6 mg Fe, 2680g carotene equivalent, 0.08 mg thiamine, 0.15 mg riboflavin, 0.5 mg niacin, and 120 mg ascorbic acid [40].

1.13 Literature Review

Many literature reports have been published demonstrating antioxidant potential of pomegranate fruit and peel. Many antioxidative compounds have been reported from these parts of pomegranate. For example:

Li.Y.et al (2009) Evaluated of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract .They found that pomegranate peel had the highest antioxidant activity among the peel, pulp and seed fractions of 28 kinds of fruits commonly consumed in China. The contents of total phenolics, flavonoids and proathocyanidins were also higher in peel extract than in pulp extract. The large amount of phenolics contained in peel extract may cause its strong antioxidant ability [41]. Maier, T.et al (2009) observed antioxidant potential of seven grape seed samples originating from mechanical seed oil extraction. The results of the present study confirm the press residues of grape seed oil production still to be a rich source of polyphenolics with strong antioxidant activity. Additionally, the effects of different solvents on the yields of phenolic compounds were determined. Maximum yields were obtained using methanol/0.1% HCl (v:v), water [75 °C] and a mixture of ethanol and water [3:1; v:v], respectively, whereas pure ethanol resulted in poor polyphenol extraction [42]. Astadi, I. R. et al (2008) measured antioxidant activity of anthocyanins of black soybean seed coat in human low density lipoprotein (LDL).they examined antioxidant activity of extract against DPPH radical and LDL oxidation. These results suggest that black soybean seed coat has high levels of phenolic and anthocyanin, and also demonstrated considerable antioxidant activity of black soybean seed coat [43]. Ng, T. B. et al (2008) examined antioxidative activity of natural products from plants. A variety of flavonoids, lignans, an alkaloid, a bisbenzyl, coumarins and terpenes isolated from Chinese herbs was tested for antioxidant activity. The flavonoids baicalin and luteolin-7-glucuronide-6′methyl ester, the lignan 4′-demethyldeoxypodophyllotoxin, the alkaloid tetrahydropalmatine, the bisbenzyl erianin and the coumarin xanthotoxol exhibited potent antioxidative activity in lipid peroxidation [44]. Luther, M. et al (2008) examined Inhibitory effect of Chardonnay and black raspberry seed extracts on lipid oxidation in fish oil and their radical scavenging and antimicrobial properties. They were also tested for radical scavenging activity against DPPH and peroxyl radicals as

reflected in oxygen radical absorbance capacity (ORAC), and total phenolic content (TPC). Both tested seed flour extracts suppressed lipid oxidation and rancidity development in fish oil. Black raspberry seed flour extract significantly reduced the degradation of biologically important n − 3 PUFA under accelerated oxidative conditions. Both seed flour extracts exhibited DPPH radical quenching activity. The data from this study suggest the potential for developing natural food preservatives from these seed flours for improving food stability, quality, safety, and consumer acceptance [45].

Zhou,K. et.al (2007) measured total phenolic contents, chelating capacities, and radicalscavenging properties of black peppercorn, nutmeg, rosehip, cinnamon and oregano leaf against cation (ABTS +), DPPH , peroxyl (ORAC) and hydroxyl (HO ) radicals. The extracts of all botanical samples showed significant radical-scavenging capacities, TPC and chelating abilities. The 50% acetone extracts of black peppercorn and cinnamon showed higher ABTS+-scavenging, ORAC, Fe+2 chelating ability and TPC value. (ESR) measurements demonstrated that cinnamon had the strongest HO -scavenging activities [46].

Maisuthisakul, P.et al (2007) obtained ethanolic extracts from various parts of 26 Thai indigenous plants and examined for phenolic constituents and free radical scavenging capacity, Total phenolic content and total flavonoid content were evaluated according to the FolinCiocalteu procedure, and a colorimetric method, respectively. The results showed that total phenolic compounds and flavonoid content were higher in seed extracts of berries used in wine production, while the levels in extracts obtained from herbs and vegetables were lower. Chewing plants which have significantly higher total phenolic content and flavonoid content [47]. Khan, M.A. et al (2007) compared the effects of natural and synthetic antioxidants on the oxidative stability of borage and evening primrose triacylglycerols. Results suggest that tocopherols are more effective antioxidants at 500 ppm than at 200 ppm. The most effective natural antioxidant was Tenox GT-2 followed by δ- and α-tocopherols, while, among synthetic antioxidants, (TBHQ) was more effective than (BHA) and (BHT) and served as the strongest antioxidant in borage and evening primrose oil TAG [48]. Nuutila, A.M.et al (2007) compared antioxidant activities of onion and garlic extracts in methanol by inhibition of lipid peroxidation and radical scavenging activity against

diphenylpicrylhydrazyl radical.They showed that onions had higher radical scavenging activities than garlic, red onion being more active than yellow onion. The skin extracts of onion possessed the highest activities [49]. Esposito, F.et al (2007) worked on antioxidant activity and dietary fibre in durum wheat bran byproducts.They investigated, two commercial products Bran & Brain 50 and 70. The antioxidant activity of some durum wheat by-product fractions is comparable to that of widespread fruits and fresh vegetables, likely due to the presence of fibre-bound phenol compounds [50]. Aguirrezabal, M. M. et al (2007) observed the effect of paprika, garlic and salt on rancidity in dry sausages. Spanish paprika and salt showed antioxidant and prooxidant properties, respectively. Paprika was even able to inhibit the prooxidant effect of salt. Also, four batches of chorizo were made to compare the antioxidant effect of the spices (garlic and paprika) with a mixture of nitrate, nitrite and ascorbic acid. In this respect, paprika and garlic were as effective as the mixture of additives in inhibiting lipid oxidation [51].

Lambropoulos, I.et al (2007) observed antioxidant activity of Xinomavro red wine phenolic extracts towards oxidation of corn oil. . One wine extract, rich in phenolic acids and flavonols, inhibited the oxidation of corn oil stripped of tocopherols to a greater extent than butylated hydroxyanisole, at 200 mg/L.Other extract, at 100 mg/L total phenolics, rich in flavanols, flavonols and tyrosol, also exhibited high inhibitory action.Results indicated that some red wine phenolics - such as phenolic acids, flavonols or flavanols - may be strong antioxidants in corn oil [52]. Iqbal, S.et al (2007) measured antioxidant properties and components of some commercially available varieties of rice bran in Pakistan. Five indigenous rice bran varieties were, i.e. Rice bran-Super kernel (RB-kr), Rice bran-Super 2000 (RB-s2), Rice bran-Super Basmati (RB-bm), Rice bran-Super-386 (RB-86) and Rice bran-Super fine (RB-sf). The overall order of antioxidant activity was RB-kr > RB-s2 > RB-bm > RB-86 > RB-sf. However, according to the chelating activity and conjugated dienes assays the antioxidant efficacy of RB-sf was higher than RB-bm and RB-86 [53]. Han,J. et.al (2007) examined antioxidants in a Chinese medicinal herb– Lithospermum erythrorhizon.They isolated Seven compounds, deoxyshikonin (1), β,β-dimethylacrylshikonin (2), isobutylshikonin (3), shikonin (4), 5,8-dihydroxy-2-(1-methoxy-4-methyl-3-pentenyl)-1,4-

naphthalenedione (5), β-sitosterol (6) and a mixture of two caffeic acid esters (7). Antioxidant activities, assessed by Rancimat method and reducing power, decreased in the following order, respectively: compound 7 > 4 > BHT > 2 > 3 > 5 > 1 > 6 [54]. Martín-Diana, A. B. et al (2007) used Green tea extract as a natural antioxidant to extend the shelf-life of fresh-cut lettuce. Optimal GT treatment (0.25 g 100 mL− 1 at 20 °C) was compared with chlorine (120 ppm at 20 °C). High GT concentrations (0.5 g 100 mL− 1 and 1.0 g 100 mL− 1) maintained better prevent ascorbic acid and carotenoid loss than 0.25 g 100 mL− 1 GT and chlorine [55]. Peschel, W.et al (2007) searched natural antioxidants from vegetable and fruit wastes. Eleven fruit and vegetable byproducts and two minor crops were screened for antioxidant activity.Extracts with the highest activity, economic justification and phenolic content were obtained from apple, pear, tomato, golden rod and artichoke. Apple, golden rod and artichoke byproducts were extracted at pilot plant scale and their antioxidant activity was confirmed by determination of their free radical scavenging activity (DPPH) and the inhibition of stimulated linoleic acid peroxidation (TBA and Rancimat® methods). This study demonstrated the possibility of recovering high amounts of phenolics with antioxidant properties from fruit and vegetable residuals not only for food but also cosmetic applications [56]. Anna,P.(2007) focused on the content, composition, and antioxidant capacity both lipid- and water-soluble antioxidants in raw Brassica vegetables, which include different genus of cabbage, broccoli, cauliflower, Brussels sprouts, and kale, are consumed all over the world [57].

Sultana,B.et al (2007) examined antioxidant potential of corncob extracts for stabilization of corn oil subjected to microwave heating.Extracts were prepared in n-hexane, ethyl acetate, acetone, ethanol, and methanol and was assessed for total phenolics content (TPC), DPPH radical scavenging activity and % inhibition of peroxidation in linoleic acid system. . Methanolic extract offered the highest yield (19.5%), and also exhibited superior antioxidant activity. The results of different antioxidant parameters investigated that corncob is a potent source of natural antioxidants that might be explored to prevent oxidation of vegetable oils [58].

Nedyalka et.al (2006) obtained natural antioxidants from herbs and spices (ground materials or extracts) and reported the structure of the main antioxidatively acting compounds isolated from them.They studied antioxidative effects of rosemary, sage, oregano, thyme, ginger, summer savory, black pepper, red pepper, clove, marjoram, basil, peppermint, spearmint, common balm, fennel, parsley, cinnamon, cumin, nutmeg, garlic, coriander, etc. Among these rosemary is extensively studied its extracts are the first marketed natural antioxidants [59].

Wong, S.P, et al (2006) examined antioxidant properties of 25 edible tropical plants using DPPH (1,1-diphenyl-2-picrylhydrazyl free radical) scavenging and reducing ferric ion antioxidant potential (FRAP) assays.They suggested that polyphenols in the extracts were partly responsible for the antioxidant activities.While TEACDPPH, (Trolox equivalent antioxidant capacity) TEACFRAP and TPC contributed to the total variation in the antioxidant activities of the plants [60].

Ozturk, S.et al (2006) examined the effect of antioxidants on butter in relation to storage temperature and duration. Natural ( -tocopherol) and synthetic (BHA and BHT) antioxidants were added to the butter samples at two concentrations (50 and 100 ppm). . Peroxide value (PV) and thiobarbituric acid (TBA) number and the residual antioxidants of the samples were examined at 30-day intervals.The use of

-tocopherol is recommended as a natural antioxidant

to suppress the development of rancidity in butter [61].

Abdalla, A.E.M. et al (2006) collected Egyptian mango seeds as wastes from local fruit processing units, the kernels were separated and dried. The antioxidan and antimicrobial activities of mango seed kernel extract and oil were investigated. The results indicated that combination of both mango seed kernel extract and oil had optimum antioxidant potency higher than each one alone [62]. Pike, P. R. et al (2006) observed antioxidant activity of oat malt extracts in accelerated corn oil oxidation.They used methanol to isolate the crude antioxidants.Antioxidant ability was compared with synthetic antioxidant butylated hydroxytoluene (BHT).Oat malt extracts showed remarkable antioxidant activity [63].

Serra, A.T. et al (2006) used olive- and grape-based natural extracts as potential preservatives for food. Results suggested that the natural extracts may have important applications in the future as natural antimicrobial agents for food industry as well as for medical use. The natural extracts showed more antimicrobial activity than shown by the selected antioxidants alone against all microorganisms [64]. Amin.I.et al. (2005) determined the total antioxidant activity and phenolic content of Kale, spinach, cabbage, swamp cabbage and shallots. Shallots showed the highest total antioxidant activity followed by spinach, swamp cabbage, cabbage and kale [65]. Descalzo, A.M. et al (2005) worked on natural antioxidants and their effects on oxidative status, odor and quality of fresh beef produced in Argentina. Pasture samples typically have higher levels of α-tocopherol, β-carotene, ascorbic acid and glutathione than feedlot samples. These compounds retard lipid and protein oxidation in fresh and stored meat, and preserve the color and odor quality of beef [66]. Wanasundara, U.N.et al (2005) detrmined the antioxidative activity of ethanolic extracts of canola meal at 100, 200, 500 and 1000 ppm on refined-bleached (RB) canola oil and compared with commonly used synthetic antioxidants, such (BHA), (BHT), BHA/BHT/monoglyceride citrate (MGC) andtert-butyl-hydroquinone (TBHQ). Stability of RB oil was monitored under Schaal oven test conditions at 65°C over a 17-d period. Progression of oxidation was monitored by weight gain, peroxide, conjugated diene, 2-thiobarbituric acid and total oxidation values. Canola extracts at 500 and 1000 ppm were more active than BHA, BHT and BHA/BHT/MGC and less effective than TBHQ at a level of 200 ppm [67]. Pinelo, M. et al (2005) took pine sawdust and almond hulls, for extraction of natural antioxidants under different experimental conditions.1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical was used for measuring antioxidants activity. Ethanol, methanol and water were used as extracting solvents.Pine sawdust offered the best results, with a 3–10 times higher (0.1122 g/100 g in dry basis) total phenolics content than almond hulls [68]. Yingming, P.et al (2004) examined antioxidant activities of several Chinese medicine herbs. The antioxidant activity (AA) of ethyl acetate extracts of Caesalpinia sappan, Lithospermum erythrorhizon, Anemarrhena asphodeloides, Paris polyphylla and Illicium verum were tested in refined peanut oil at 60 ± 0.5 °C. All of C. sappan, L. erythrorhizon extracts and their combinations were found to be high effective in peanut oil. But the extracts of A. asphodeloides,

P. polyphylla and I. verum slightly decrease the formation of peroxides in peanut oil as compared with pure oil [69]. Gramza.A.et al (2004) studied Tea constituents (Camellia sinensis L.) as antioxidants in lipid systems.They concluded that tea contain polyphenols which act as antioxidants [70].

Batifoulier, F. et al (2004) isolated a novel type of antioxidant from leaf wax of Eucalyptus globulus leaves and identified as n-tritriacontan-16, 18-dione. The antioxidant showed remarkable antioxidative activity in a water/alcohol system and was more effective than αtocopherol and BHA [71]. Yin, et. al (2004) examined nonenzymatic Antioxidant Activity of Four Organosulfur Compounds Derived from Garlic.The compounds were diallyl sulfide (DAS), diallyl disulfide (DADS), S-ethyl cysteine (SEC), and N-acetyl cysteine (NAC). On the basis of the observed nonenzymatic antioxidant protection, these organosulfur compounds are potent agents for enhancing lipid stability [72]. Iqbal,S.et al (2004) examined antioxidant properties and components of bran extracts from selected wheat varieties commercially available in Pakistan.They slected five wheat varieties indigenous to Pakistan, i.e. Punjab-96, Bhakkar-2002, Uqab-2000, SH-2002, and Pasban-90. All the varieties exhibited appreciable antioxidant potential and significant differences were observed among the varieties in different systems of antioxidant activity evaluation [73]. Desmarchelier, C et.al (2003) studied antioxidant and free radical scavenging properties of bark extracts of Anadenanthera macrocarpa Brenan (Fabaceae), Astronium urundeuva Engl. (Anacardiaceae), Mimosa verrucosa Benth. (Fabaceae) and Sideroxylon obtusifolium T.D. Penn. (Sapotaceae).They used aqueous and methanolic extracts.They tested antioxidant activity on 2,2′-azo-bis(2-amidinopropane) as a peroxyl radical source. The highest activity was observed in the methanolic extract of A. macrocarpa (TRAP=3028±95 μM) [74]. Batifoulier, F. et al (2003) observed Influence of vitamin E on lipid and protein oxidation in microsomal membranes from turkey muscle. Lipid oxidation was estimated by TBARS and protein oxidation was measured by an estimation of carbonyl groups and free thiols. Vitamin E protected free thiols from oxidation but had only a small effect Vitamin E supplementation significantly protected free thiols from oxidation but had only a small effect (non significant) on carbonyl group formation.on carbonyl group formation [75].

Litridou M.et al (2003) fractionated phenolic compounds in olive oils and checked their antioxidants activity. The polar fraction of virgin olive oil was separated into two main parts (A and B) using solid phase extraction. The two parts tested for their antioxidant activity showed relatively high protection factors in safflower oil stored at 80°C. Part B was found to contribute more than part A to the stability of the oil [76]. Xing, Y.et al (2003) identified and quantified methanolic extracts of groats and hulls from Ogle oat by usings GC-MS. Extracts from groats and hulls at levels of 0.05, 0.1, 0.2, and 0.3% w/w, based on total phenolic content, were added to soybean oil, and their antioxidant effectiveness was compared with that of 0.02% w/w tertiary butylhydroquinone (TBHQ) by measuring peroxide values. During 20 d of storage, the groat extract (0.3%) was not significantly different from TBHQ after day 16, and hull extracts (0.2 and 0.3%) were not significantly different from TBHQ on day 20 [77]. Duh, P.D.et al (2003) studied antioxidant efficacy of methanolic extracts of peanut hulls in soybean and peanut oils. . Results showed that the oils with 0.12, 0.48, and 1.20% MEPH had significantly (P<0.05) lower peroxide values and acid values than the control after storage at 60°C. . Moreover, oils with 0.48 and 1.20% MEPH were significantly (P<0.05) superior to 0.02% butylated hydroxyanisole (BHA) in reducing oxidation of both oils [78]. Desmarchelier, C et al (2002) revived the advances in the search for antioxidant activity in plants used as medicinal agents in different areas of South America [79]. Wanasandara, U. N et.al (2002) stabilized canola oil with flavonoids. Results were compared with, BHA and BHT. Oxidation was monitored by weight gain, peroxide value (PV) and 2thiobarbituric acid reactive substances (TBARS) value.Among the flavonoids tested, myricetin,epicatechin, naringin, rutin, morin, and quercetin were superior to BHA and BHT in inhibiting oil oxidation [80]. McCarthy,T.L.et al.(2001) examined the antioxidant potential of aloe vera (AV), fenugreek (FGK), ginseng (G), mustard (M), rosemary (R), sage (S), soya protein (SPI), tea catechins (TC) and whey protein concentrate (WPC) in raw and cooked patties manufactured from frozen pork.They were AV (0.25%), FGK (0.01%), G (0.25%), M (0.10%), R (0.10%), S (0.05%), SPI (0.10%), TC (0.25%) and WPC (4%).While for BHT and BHA they were (0.01%).They concluded that ranking of decreasing antioxidant effectiveness is Control>G>SPI>FGK>AV>M>WPC>S>α-tocopherol>R>TC>BHA/BHT [81].

Wanasundara, U. N.et al (2001) stabilized seal blubber and menhaden oils with green tea catechins. The antioxidant activity of isolated catechins was compared with those of αtocopherol, (BHA), (BHT), (TBHQ), all at 200 ppm. Oils treated with tea catechins showed excellent oxidative stability as compared with samples that contained commonly used antioxidants [82]. Amarowicz, R. et.al (2001) examined free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Ethanolic extracts from the roots of wild licorice (Glycyrrhiza lepidota), narrow-leaved echinacea (Echinacea angustifolia), senega (Polygala senega), leaves of bearberry (Arctostaphylos uva-ursi) and aerial parts of two varieties of horsetail (Equisetum spp.) were prepared and evaluated for their free-radical scavenging capacity and their antioxidant activity. The bearberry-leaf extract consistently exhibited the highest antioxidant activity based on the tests performed, and seems to be a promising source of natural antioxidants [83]. Pokorny, J. et. al (2000) obtained natural antioxidants from herbs and spices and their effect on the keepability of foods [84]. Farag R. S. et al (1999) examined Influence of thyme and clove essential oils on cottonseed oil oxidation. Three methods were used to follow cottonseed oil oxidation, i.e., coupled oxidation withβ-carotene, the TBA test and hydroperoxide number. The results illustrated that clove and thyme oils at various concentrations exhibit antioxidant activity and this phenomenon for clove oil is superior to that of thyme oil [85]. George, B.et al (1999) studied bio-antioxidant content and antioxidant activity of 12 tomato genotypes. Significant differences were found between lycopene, ascorbic acid and phenolic contents among various genotypes [86]. Mohamed H. M. A et al (1999) isolated sesame oil unsaponifiable matter from two different coloured seed varieties (white and brown). The brown variety contained higher amounts of total sterols and tocopherols but lower amounts of sesamin, sesamolin than the white variety.Both were added in sunflower oils and their effectiveness was compared with a control (no additives) at 63 °C. Results indicated that both had antioxidant activity which increased with increasing concentration [87].

Kang, M.H. et al (1999) used sesame lignans in protecting lowdensity lipoprotein against oxidative damage. Sesaminol inhibited the Cu2+-induced lipid peroxidation in LDL. Sesaminol was a more effective scavenger than either α-tocopherol or probucol in reducing the peroxyl radicals [88].

Osawa, T (1999) reviewed functions of dietary antioxidants which were obtained from foods [89]. Chen, X.et al (1998) noticed antioxidant activities of six natural phenolics against lipid oxidation induced by Fe2+ or ultraviolet light.They used thiobarbituric acid-reactive substances (TBARS) method to determine lipid oxidation. The antioxidant activities of the six phenolics against UVinduced lipid oxidation were as follows: quercetin > rutin = caffeic acid = ferulic acid = sesamol > catechin.And against Fe2+-induced lipid oxidation was in the order quercetin (1.7 µM) > rutin (10.3 µM) > catechin (14.9 µM) > sesamol (18.5 µM) > caffeic acid (19 µM) > ferulic acid (>250 µM).Quercetin was more efficient than butylated hydroxytoluene (BHT) (2.9 µM) [90]. Shahid.S.A.et al (1998) examined antioxidant activity of different solvent extracts of rice bran at accelerated storage of sunflower oil. The antioxidant activity of different extracts of rice bran (var. Super Kernel) prepared using a number of solvents (100% methanol, 80% methanol, 100% acetone and 80% acetone) were evaluated in sunflower oil (SFO) under accelerated storage conditions. The overall order of antioxidant efficacy of rice bran extracts as determined by various antioxidant assays was 80% methanolic extract, >100% methanolic extract, >80% acetone extract and >100% acetone extract [91]. Edwin N.et al (1998) reported antioxidants in lipid foods and their impact on food quality.They observed that tocopherols and ascorbic acid are present in edible oils. These antioxidants can interrupt lipid autoxidation by interfering with either the chain propagation or the decomposition processes [92].

Ganthavorn, C.et al (1997) inhibited of soybean oil oxidation by extracts of dry beans (Phaseolus vulgaris). Polyphenolic compounds were extracted from pinto, kidney, white (Great Northern), pink, and black beans by hot methanol extraction and added to soybean oil. Oil oxidation was assayed by (TBARS). Bean extracts effectively inhibited iron-catalyzed oxidation of soybean oil [93].

Bin, Z.et al (1997) examined antioxidant activity of raisin extracts in bulk oil, oil in water emulsion, and sunflower butter model systems. Peroxide values and hexanal content were measured on a half day, 2 or 3 day basis for the emulsion, sunflower butter, and bulk oil, respectively. The RE had the best antioxidant activity in the bulk oil system [94]. Suja, K. P. et al (1997) observed antioxidant efficacy of sesame cake extract in vegetable oil protection. Antioxidant activity of methanolic extract of sesame cake was evaluated in soybean, sunflower, and safflower oils, using the Schaal oven method and differential scanning calorimetry (DSC) analysis. Results showed that sesame cake extract (SCE), at concentrations of 5, 10, 50 and 100 ppm in vegetable oils, could significantly (P<0.05) lower the peroxide value, diene value and p-anisidine value of oils during storage at 60 °C. The study also indicated a better antioxidant effect for sesame cake extract than BHT at 200 ppm [95]. 56. Marinova, E.M. et al (1997) observed antioxidative properties of syringic, 3,4dihydroxybenzoic, sinapic and caffeic acids in the concentration range 0.002–0.02% during autoxidation of triacylglycerols of sunflower oil at 22 and at 90 °C. The effectiveness of the phenolic acids increased in the following order: syringic acid <3,4-dihydroxybenzoic acid <sinapic acid
compounds effectively inhibited conjugated diene hydroperoxide formation in corn oil, soya bean oil, peanut oil and fish oil, when tested in bulk [99]. Chen, H-Y.et al (1994) evalouted antioxidant activity of aqueous extracts of some selected nutraceutical herbs namely Psidium guajava L. (PE), Camellia sinensis (GABA tea; CE), Toona sinensis Roem. (TE) and Rosemarinus officinalis L. (RE). Among the four extracts, PE exhibited the strongest efficiency.The reducing power of four nutraceutical herbs was in the order of PE > RE > CE > TE. These results show that the tested herbal tea, especially PE could be considered as a natural antioxidant source [100]. Edwin N.et al (1993) showed that flavonoids are an important part of the diet because they can modulate lipid peroxidation involved in atherogenesis, thrombosis, and carcinogenesis. Known properties of flavonoids include free radical scavenging, strong antioxidant activities in preventing the oxidation of low-density lipoproteins [101].

Edwin N.et al (1993) reported methods of determining the oxidative stability of food lipids and the effectiveness of natural antioxidants [102].

1.14 Aims and Objectives of Work Main objectives of the study are as follows: 1. To quantify the total phenolic content, from brassica leaves extracts. 2. To investigate the antioxidant potential of brassica leaves extracts in linoleic acid model system. 3. To evaluate the reducing power and chelating activity of brassica napus extracts. 4. Assessment of DPPH• scavenging ability. 5. To draw logical conclusion about antioxidant potential of brassica napus extracts. 6. To compare the antioxidative efficiency of brassica napus extract with synthetic antioxidants such as BHA/BHT in stabilization of sunflower oil under accelerated storage conditions.

1.15

Scope of Work/Study

With the advent of industrial revolution, environmental pollution has increased significantly. As a result, chances of cardiovascular diseases and cancer have increased. Many epidemiological studies have suggested that antioxidative compounds from different plant sources are useful in the control of these diseases. Many plant polyphenols, such as ellagic acid, catechins, chlorogenic, caffeic and ferulic acids, as well as their dietary sources, such as tea, have been shown to act as potent antimutagenic and anticarcinogenic agents [103].

Vegetable oils now-a-days are a great source of balancing oil consumption in families and because of consumers concern with the saturated/unsaturated fatty acid ratio in the diet, the lipid composition of fruit and vegetable has lately received particular attention. Consumers are especially interested in essential fatty acids, with emphasis on the health potential of polyunsaturated fatty acids. It is considered that these fatty acids play a natural preventive role in cardiovascular diseases and in alleviation of some other health problem, because they promote the reduction of both total and HDL cholesterol [104]. But these fatty acids are damaged by oxidation process and shelf life of oils is decreased due to lipid oxidation/rancidity. Synthetic antioxidants as additives into the oils are not only expensive, but also carcinogenic. So now-adays focus of research is to find antioxidants from natural sources. In the last few years, an increased attention has been focused on the industrial wastes, especially those containing residual phenols from the plant raw material used [105]. Brassica is cultivated in upper Punjab and its seeds are used for oils while its leaves are used as animal food. This research project follows a line of investigation on the antioxidative potential from brassica leaves and its efficacy in stabilization of sunflower oil under accelerated storage conditions. CHAPTER 2

MATERIALS AND METHODS 2.1 Samples Fresh leaves of brassica napus were collected from agricultural plots in polyethylene bags. These bags were made air tight and stored at 40C in a cooler.

2.2 Chemicals and Reagents

All reagents (analytical and HPLC) used were produced from E Merck or Sigma-Aldrich unless stated otherwise.

2.3 Drying and Grinding Brassica napus leaves were placed in diffused sunlight for 20 days until they were totally dried. Then they were grinded to fine powder form in grinder.

2.5 Extraction of Total Antioxidants Extraction was carried out following the method reported by Zuo, Chen and Deng (2002). Leaves samples were ground to pass 1-mm sieve and extracted thrice with 25 ml of 80% methanol for 3 h in an electrical shaker at room temperature. The contents of the flasks were further extracted twice with 20 ml of 80% methanol containing 0.15% HCl under the same set of conditions. The extracts were combined and filtered through a 0.45 µm of Nylon membrane filter. The extracted were evaporated to dryness under reduced pressure at 450 C by a rotary vacuum evaporator and stored under freezer at -18oC until used for further analysis.

2.6 DPPH• Scavenging Assay , Free radical scavenging activities of leaves extracts was determined by using a stable 2,2 -

.

diphenyl-1-picrylhydrazyl radical (DPPH ) following a previously reported method(Heinonen, Lehtonen and Hopea,1998). Briefly, a freshly prepared solution of DPPH. (5.0ml) was added to 1.0 ml of bran extracts. The decrease in absorbance, measured at different intervals, i.e.0, 0.5, 1, 2, 5, and 10 min ( up to 50%) at 515 nm was determined with a Hitachi UV-Vis model U-2000 spectrophotometer. The remaining concentration of DPPH. In the reaction medium was calculated from a standarad calibration curve. The absorbance measured at 5 min of the antioxidant-DPPH radical reaction was used to compare the DPPH radical scavenging capacity of each leaves extracts.

2.7 Determination of Total Phenolic Contents (TPC)

The total phenolic content of leaves extracts was determined using the Folin-Ciocalteu reagent (Osawa and Namiki, 1981; Singleton and Rossi, 1951). The reaction mixture contained 200 µl of diluted leaves extracts, 800 µl of freshly prepared diluted Folin-Ciocalteu reagent and 2 ml of 7.5% sodium carbonate. The final mixture was diluted to 7 ml with deionized water. Mixtures were kept in dark at ambient conditions for 2 h to complete the reaction. Then the absorbance at 765 nm was measured on Perkin-Elmer Lambda-2 Spectrophotometer, with a 1 cm cell. Galic acid was used as a standard and results were calculated as gallic acid equivalents (g/1oog) of leaves. The reaction was conducted in triplicate and results were averaged.

2.8 Chelating Activity Fe 2+ chelatiog activity was measured by a 2,2,-bipyridyl competition assay(Re et al, 1999). The reaction mixture contained 0.25ml of 1mM FeSO4, 1ml of Tris-HCl buffer (pH 7.4), 0.25ml of extract, 0.4 ml of 10% hydroxylamine-HCl, 1ml of 2,2.-bipyridyl solution (0.1% in 0.2 M HCl) and 2.5 ml of ethanol. The final volume was made up to 6.0 ml with water. The absorbance at 522 nm was meadured and used to evaluate ethylenediaminetetracetate (Na2EDTA

2.9 Antioxidants Activity Determination in Linoleic Acid System The antioxidant activity of sample extracts was determined following a reported method of Osawa and Namiki (1981). Sample extracts were added to solution mixture of linoleic acid (0.13ml), 99.8% ethanol (10ml), and 0.2M sodium phosphaste buffer, (pH 7.0, 10). The total volume was adjusted to 25 ml with disstiled water. The solution was incubated at 40oc and the degree of oxidation was measured according to the thiocyanate method (Yen, Duh and Tsai,1993) with 10 ml ethanol (75%), 0.2 ml of an aqueous solution of ammonium thiocyanate (30%), 0.20 ml sample solution and 0.20 ml of ferrous chloride(FeCl2) solution (20mM in 3.5 % HCl) being added sequentially. After 3 minute of stirring the absorption values of mixtures measured at 500 nm were taken as peroxide contents. A control was performed with linoleic acid but without the extracts. Synthetic antioxidants, BHT and α-tocopherol were used as positive control. The percent inhibition of linoleic acid peroxidation, 100-[(Abs. increase of sample at 360h/Abs. Increase of control at 360h)×100], was evaluated to express antioxidative activity.

CHAPTER 3 RESULTS AND DISCUSSION 3.1 DPPH Radical Scavenging Abilities DPPH is a stable free radical and has been widely used to evaluate the free radical scavenging ability of various botanical materials [106]. This method presents the advantage of using a stable and commercially available free radical and has been extensively applied on the study of antioxidant activity of food items, such as olive oil, fruits, juices and wines [107]. The DPPH• free radical has been used to assess the ability of phenolic compounds to transfer labile “H” atoms to radicals [108]. This method permits to evaluate not only the electron or hydrogen atom donating properties of antioxidants, but also the rate of their reaction towards the free radicals. Results for free radical scavenging capacity of Brassica napus were recorded as remaining amount % of DPPH• after 7 minute of mixing of DPPH• with brassica napus extract. The results were (64.12±2.07). These results are higher than those onion (31.1 ± 2.0) potato peels (33.5 ± 2.7) and wheat bran (30.1 ± 3.3) [109].

3.2 Total Phenolic Content (TPC)

Model studies have demonstrated that most of the phenolics possess antioxidant activity. The antioxidant activity of phenolics is mainly because of their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators [110]. A number of studies have focused on the biological activities of phenolic compounds, which are potential antioxidants and free radical scavengers [111]. Total phenolic content (TPC) was determined following a modified Folin-Ciocalteu reagent method and results were expressed as gallic acid equivalents. TPC was 1.49 ±0.03 mg/g brassica napus extracts. Total phenolic contents were higher than Pear (peeled) (Pyris communis) 0. 91 mg/g, Apple, yellow (unpeeled) (Malus pumila ) 0.99 mg/g, Peach( Prunus persica) 0.50 mg/g [112].

3.3 Chelating Activity

Iron is an extremely reactive metal and will catalyze oxidative changes in lipids, proteins and other cellular compounds [108]. The chelating activity was measured against Fe2+and reported as EDTA equivalents. Phenolic extract of Brassica napus

showed chelating activity in range of

(549.6±2.3) µg/g. The results were comparable as those of rice bran extract (RB-bm) i.e. (698±8.2) µg/g [113].

3.4 Antioxidant Activity in Linoleic Acid System Antioxidant activity of phenolic extracts of brassica napus was observed in linolic acid system and compared with BHA and α tocopherol. The results are (36.15±1.52) % of antioxidant activity. The findings are in agreement with previously determined % antioxidant activity in garlic methanolic extracts which were (93.2±7) % [114].

3.5 Total Flavonoid Contents (TFC) Flavonoids are polyphenolic compounds that are ubiquitous in nature, found in fruits, vegetables, and certain beverages and have diverse beneficial biochemical and antioxidant properties [115]. Flavonoids provide protection against cancer and carcinogenesis through inhibition of oxidative damage. Flavonoids have been labeled as “high level” natural antioxidants on the basis of their ability to scavenge free radicals and reactive oxygen species [116,117]. Total flavonoid content in extracts from leaves of Brassica napus were (58.7±2.8).TFC of Brassica napus was found to be significantly (p < 0.05) higher than that of flavonoid content from apple peels (3.06 mg/g) [118], which have been reported as potential source of antioxidants. The Brassica napus exhibited the highest antioxidant activity, perhaps due to the reducing power of flavonoids.

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