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Lour.), NUTMEG (Myristica fragrans) AND SNAKE FRUIT (Salacca edulis Reins.) SEEDS.





In preparing this project, I was in contact with many people, academicians and practitioners. They have contributed times and knowledge towards my understanding and thoughts. In particular, I would like to express my sincere appreciation to my main project supervisor, Madam Suzaira bt Bakar for her encouragement, support, guidance, critics and friendship. Without her continued support and interest, this project would not have been the same as presented here. Not forget to my programme mates who support, help and encourage me to finish my project on time. Not forgetting to my lovely parents who are supporting me in mental and financial while doing this project. Finally, thanks to all my lectures of Food Science and Technology in Faculty of Applied Sciences and to anyone who was not mention above, whom indirectly gave me an advice, support, ideas and all sorts of things that help me finish this project.

Farhana binti Mohamed Wazir


TABLE OF CONTENTS Page ACKNOWLEDGEMENTS TABLE OF CONTENT LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ABSTRACT ABSTRAK CHAPTER 1 INTRODUCTION 1.1 Background and problem statement 1.2 Significance of study 1.3 Objectives of study CHAPTER 2 LITERATURE REVIEW 2.1 Nutmeg (Myristica fragrans) 2.1.1 Illustration taxanomy 2.1.2 General information 2.1.3 Pharmacologically active parts of the plant 2.1.4 Chemical composition 2.1.5 Antioxidant properties of nutmeg 2.2 Longan (Dimocarpus longan) 2.2.1 Illustration taxanomy 2.2.2 General information 2.2.3 Antioxidant properties of longan 2.2.4 Food exploitation 2.3 Snake fruit (Salacca edulis Reinw.) 2.3.1 Illustration taxanomy 2.3.2 General information 2.3.3 Antioxidant properties of snake fruit 2.3.4 Food exploitation 2.4 Antioxidants 2.4.1 Classification of antioxidant based on mechanism of action 2.4.2 Free Radical 2.4.3 Flavonoid Compound 2.4.4 Ascorbic acid 2.4.5 Phenolic compound 2.4.6 Synthetic antioxidant iii iv vi vii viii ix x

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CHAPTER 3 METHODOLOGY 3.1 Materials 3.1.2 Samples 3.1.3 Chemicals 3.14 Equipment iv COPYRIGHT UiTM

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Methods 3.2.1 Preparation of fresh sample Longan (Dimocarpus longan) Snake fruit (Salacca edulis Reinw) Nutmeg (Myristica fragnans) 3.2.3 Preparation of cooked sample Longan (Dimocarpus longan) Snake fruit (Salacca edulis Reinw) Nutmeg (Myristica fragnans) 3.2.4 Extraction of plant sample 3.2.2 Antioxidants test Total flavonoid content Total phenolic content Total ascorbic acid 3.2.6 Statistical analysis

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CHAPTER 4 RESULTS AND DISCUSSION 4.1 Total flavonoid content 4.2 Total phenolic content 4.3 Total ascorbic acid content 4.4 Correlation between total phenolic content with total flavonoid content CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion 5.2 Recommendations

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LIST OF TABLES Table 4.1 Caption Total flavonoid content in fresh and cooked longan, nutmeg and snake fruit 4.2 Total phenolic content in fresh and cooked longan, nutmeg and snake fruit 4.3 4.4 Ascorbic acid content in fresh and cooked longan, nutmeg and snake fruit Correlation coefficients between total flavonoid content and total phenolic content in fresh and cooked longan, nutmeg and snake fruit. 30 33 28 Page 26


LIST OF FIGURES Figure 2.1 2.2 2.3 2.4 2.5 2.6 3.1 4.1 4.2 4.3 Caption Nutmeg seeds Seeds of longan Snake fruit pulp and seeds Basic chemical structure of antioxidants Cause and effect of free radicals Basic flavanoid structure Flowchart of preparation of seed extracts Graph of flavonoid content Graph of phenolic content Graph of ascorbic acid content Page 5 8 11 13 15 16 21 27 29 31


LIST OF ABBREVIATIONS AOAC ANOVA AlCl3 CH3COOH DCIP GAE HPO3 NaNO2 Na2CO3 RE mL cm C % g wt M mg/L rpm SPSS : : : : : : : : : : : : : : : : : : : : Association of Official Analytical Chemists Analysis of Variance Aluminium Chloride Acetic Acid 2, 6-dichloroindophenol Gallic Acid Equivalent Metaphosphoric Acid Sodium Nitrate Sodium Bicarbonate Rutin Equivalent miliLitre centimeter degree Celcius percent gram weight Molarity milligram per Litre rotation per minutes Statistical Package for Social Sciences



The aim of this study is to determine the content of total flavonoid, phenolic and ascorbic acid content in six samples which are fresh and cooked longan, nutmeg, and snake fruit seeds. This study was carried out to observe the effects of cooking on total flavonoid, phenolic content and ascorbic acid in six samples. Before the analysis, all fresh and cooked samples were extracted by using ethanol to obtain extracts of sample for total flavonoid and phenolic content. For ascorbic acid test, all samples were homogenized with metaphophoric acid-acetic acid solution. The total flavonoid content were determined by using total flavonoid content method at 510 nm against a standard curve with rutin as a standard. The highest amount of total flavonoid content was in fresh longan seeds with 715.19 3.56 mg RE/100g and the lowest amount of flavonoid content was in cooked snake fruit seeds with 163.99 6.58 mg RE/100g. The total phenolic content were determined by using total phenolic content method through direct spectrophotometric absorption at 750 nm against a standard curve with gallic acid as a standard. The highest amount of total phenolic content was in fresh longan seeds with 952.60 5.84 mg GAE/100g and the lowest amount of phenolic content was in cooked snake fruit seeds with 277.24 2.68 mg GAE/100g. Ascorbic acid content was determined by using AOAC method. The highest amount of ascorbic acid content was fresh longan seeds with 118.375 5.92 mg ascorbic acid/100g and the lowest amount of ascorbic acid was cooked snake fruit seeds with 44.686 2.25 mg ascorbic acid/100g. All samples are significant differences between fresh and cooked samples at (p < 0.05). Cooked samples shown lower amount of total flavonoid, phenolic and ascorbic acid compared to the fresh samples because nutrients may lost through oxidation, especially during cooking or heating. This is more easily happened to ascorbic acid because it very sensitive to heat. It is also because decreases in synthesis or increases in degradation.



Kajian ini dijalankan adalah untuk menentukan kandungan flavonoid, fenolik dan asid askorbik di dalam enam sampel iaitu biji longan, pala, salak segar dan yang telah di masak. Kesemua sampel telah diekstrak dengan menggunakan ethanol untuk mendapatkan ekstrak fenolik dan flavonoid sebelum analisis dijalankan. Bagi kandungan asid askorbik, kesemua sampel telah dicampurkan dengan menggunakan asid metafosforik-asid asetik. Kandungan flavonoid ditentukan dengan menggunakan kaedah flavonoid yang menggunakan rutin sebagai piawaian. Jumlah flavonoid paling banyak adalah pada biji longan segar iaitu sebanyak 715.19 3.56 mg RE/100 g dan jumlah flavonoid yang paling sedikit iaitu biji salak yang dimasak sebanyak 163.99 6.58 mg RE/100 g sampel. Kandungan fenolik ditentukan dengan menggunakan kaedah fenolik yang menggunakan asid galik sebagai piawaian. Jumlah fenolik paling banyak adalah biji longan segar iaitu sebanyak 952.60 5.84 mg GAE/100 g sampel dan jumlah fenolik paling sedikit adalah biji salak yang dimasak iaitu sebanyak 277.24 2.68 mg GAE/100 g sampel. Kandungan asid askorbik ditentukan dengan menggunakan kaedah AOAC. Jumlah asid askorbik paling banyak ditunjukkan pada biji longan segar dengan 118.375 5.92 mg askorbic asid/100 g sampel untuk biji longan segar dan jumlah asid askorbik paling sedikit adalah biji salak yang dimasak iaitu sebanyak 44.686 2.25 mg ascorbic acid/100 g sampel. Kesemua sampel mempunyai perbezaan signifikan di antara sampel segar dan yang di masak pada (p < 0.05). Sampel yang telah di masak menunjukkan jumlah keseluruhan flavonoid, fenolik dan askorbik asid lebih rendah berbanding sampel segar kerana nutrien boleh hilang melalui osidasi terutamanya sewaktu masak atau di panaskan. Ini lebih mudah berlaku kepada askorbik asid kerana ia lebih sensitif kepada haba. Ia juga kerana penurunan dalam sintesis atau peningkatan dalam degradasi.





Background and problem statement Antioxidant components are microconstituents present in the diet. It can delay or inhibit lipid peroxidation by inhibiting the initiation or propagation of oxidising chain reactions. It also involved in scavenging free radicals. Food such as honey, fruits, vegetables and grains are reported to contain a wide variety of antioxidant components, including some vitamins, as well as L-ascorbic acid, phenolic compounds and others. These compounds are found to be well correlated with antioxidant activity (Katalinic et al., 2004). According to Michels et al., (2000), incidence of degenerative diseases including cancer, heart disease, inflammation, arthritis, immune system decline, brain dysfunction and cataracts can be lowered by associated with a high consumption of fruits and vegetables. Many researchers believe that vitamin supplements can reduce free radical damage. It also prevents and delays the chronic degenerative diseases, and also possibly extends lifespan (Jacob and Sotoudeh, 2002). Besides that, the synergistic effect which could exist between different antioxidants means that the total antioxidant effect may be greater than the sum of the individual antioxidant activities and the isolation of one compound will not exactly reflect the overall action (Jia et al., 1998).

Antioxidants can act as a protection agent which is able to terminate the initiation of oxidizing chain reactions. It also can inhibit or delay oxidation by other molecules (Suchandra et al., 2007). Oxidation processes are important because it can control the production of free radicals and the unbalanced mechanism of antioxidant protection that can cause diseases and 1 COPYRIGHT UiTM

accelerated ageing (Dawidowicz et al., 2006). Free radicals can also initiate the oxidation of biomolecules which will lead into cell injury and death (Freidovich, 1999).

The dietary intake of antioxidants is an important role in the protection of the human organism against free radicals. Many studies show the connection between the antioxidant activity of the substances present in the diet and the prevention from diseases such as cardiovascular diseases or carcinogenesis (Kris-Etherton et al. 2002). Free radicals play an important role in affecting human health by causing several diseases including cancer, hypertension, heart attack and diabetes. There are generated during body metabolism. Exogenous intake of antioxidants can help the body scavenge free radicals effectively. These effects have been attributed to antioxidant components such as plant phenolics, flavonoids and phenylpropanoids (Rice-Evans et al., 1996). Antioxidants also can neutralise chemically active products of metabolism. Sources of natural antioxidants are primarily phenolics that may occur in all products and parts of a plant such as fruits, vegetables, nuts, seeds, leaves, roots, and bark.

Many studies have shown that the increased dietary intake of natural phenolics can reduced coronary heart disease and cancer mortality with longer life expectancy (Halliwell, 2007). Moreover, these polyphenolic compounds have been found effective in many health-related properties, such as antioxidant, anticancer, antiviral and anti-inflammatory activities (Amin et al., 2006). On the other hand, concern about safety of the common used of synthetic antioxidants such as butylated hydroxyanisole (BHA) and tertiary butylhydroquinone (TBHQ) have led to increase the interest on consumption of natural antioxidants which occur in plants as secondary metabolites. The synthetic antioxidant which is commonly used in processed foods has some unwanted side effects and some also are carcinogenic to human being.



Significance of study This study was proposed to investigate the natural activities of longan, nutmeg and snake fruit seeds by methanol and water extraction. It would give an opportunity on ways to replace the synthetic antioxidant in the processed foods, since synthetic antioxidant is not healthy.

Fruits and vegetables consumption have been attributed in many epidemiological studies to reduce cancer and cardiovascular disease. Recent cancer literatures have focused on antioxidant supplementation during chemotherapy and found that with supplementation, there was a significant in higher survival rate, higher tumour response, fewer toxicities and increased chemotherapy efficacy (Block et al., 2007).

Malaysia has many types of fruits and vegetables. Malaysians are not interested in trying a new fruits and vegetables that are not being commercialised yet or called as indigenous fruits and vegetables. Most of these fruit and vegetable are usually found in the rural area. Because of this, there are not expose to the outsider. But for the people that live in the rural area, they are familiar with some of the fruits or vegetables that are not in the market yet because of their daily consumption of that food.

There is a growing interest in the use of natural antioxidants for expanding the shelf life of food without the need of synthetic antioxidants. Examples of synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertiary butyhydroquinone (TBHQ). These foods additive have been used to prevent lipid peroxidation that may possess possible toxic and carcinogenic effect on health (Ito et al., 1985). Thus, efforts have been made to search for novel natural antioxidants from tea, fruits, vegetables, herb and spices and by product such as skin and seeds.

The replacement of synthetic antioxidants by natural antioxidant may have benefits due to health implications and functionality. However, some of them such as those from spices and herbs have limited applications in spite of their high antioxidant activity. Naturally occurring antioxidant substances also 3 COPYRIGHT UiTM

need safety testing. Caution regarding an assumption of safety of natural antioxidants has been repeatedly advised, since the fact than an antioxidant comes from a natural source does not prove its safety.


Objectives The objectives of this study are as follows: i. to determine the content of total flavonoid, phenolic and ascorbic acid of fresh and cooked nutmeg, snake fruit and longan seeds. ii. to observe the effects of cooking on total flavonoid, phenolic and ascorbic acid content of nutmeg, snake fruit and longan seeds compare to fresh.





Nutmeg (Myristica fragrans)

Figure 2.1 Nutmeg seeds Source: Rudgley, (1998)


Illustration taxanomy Kingdom Division Class Order Species Family Genus : Plantae : Magnoliophyta : Magnoliopsida : Magnoliales : Myristica : Myristicaceae : Myristica

Source: Anon, (1995)


2.1.2 General information Nutmeg seed is grayish-brown colour. It is a wrinkled kernel of the fruit of the Myristica fragrens Houtt tree. It can be found in spherical and oval shape. Both of them appear hard, but are easily grated. When the kernel is cut transversely, many dark brown veins, containing the volatile oils, become visible. Nutmeg is in the category of tree. This tree is indigenous to the Banda islands in the Moluccas. Nutmeg is the species of the genus Myristica. It have distributed from India and South-East Asia to North Australia and the Pacific Islands.

Nutmeg tree can reach a height of 4 to 10 metres. This tree is dioecious with male and female flowers occurring on different trees. It is obligatory cross pollinated an ant mimicking flower beetle and effective pollinator in South India (Armstrong and Drummond, 1986). The fruits of this tree are pendulous, broadly pyriform, yellow and smooth. The fruits can be 710 cm long. Its flesh is split open into two halves when ripe, showing the ovoid 23 cm long dark brown shining seed with hard seed coat, surrounded by a lanciate red aril attached to the base of the seed. The seed of nutmeg is large with ruminate endosperm and is considered as the most primitive among the flowering plants (Corner, 1976).


Pharmacologically active parts of the plant The most important part of the plant in terms of its pharmacological activity and commerce is of course the dried kernel which is seed. Intoxication from the use of the aril of the fruit, generally known as mace, has also been reported, but rare. The oil of nutmeg has also been used for medicinal purposes. This is because nutmeg contains the pharmacologically active components. It is also used as a spice in various dishes, as components of tea and soft drinks or mixed in milk and alcohol. Sometimes nutmeg is used as a stomachic. It is used in traditional medicine. It is also used as a stimulant, carminative as well as for intestinal catarrh and colic, to stimulate appetite, to control flatulence and it has a reputation as an emmenagogue and abortifacient (Nadkarni, 1988).


2.1.4 Chemical composition The main constituents of M. fragrans have been found to be alkyl benzene derivatives such as myristicin, elemicin and safrole, terpenes, alpha-pinene, beta-pinene, myristic acid and trimyristin (Yang et al., 2008). Nutmeg contains about 10% essential oil. It is mostly composed of terpene hydrocarb, terpene derivatives and phenylpropanoids. Of the latter group, myristicin is responsible for the hallucinogenic effect of nutmeg. Oil of mace contains the same aroma components but the total fraction of terpenoids is increased to almost 90%. Nutmeg contains about 2% of lignans, which are non volatile dimers of phenylpropanoid constituents of the essential oil for example is dehydrodiisoeugenol (Anonymous, 1995). The main glycoside is trimyristin having anxiogenic activity (Sonavane et al., 2002).


Antioxidant properties of nutmeg Murcia et al., (2004) has carried out a study on the antioxidant properties of some spices and compared with those of the common food antioxidants butylated hydroxyanisole (BHA) (E-320), butylated hydroxytoluene (BHT) (E-321) and propylgallate (E-310). From the results, nutmeg showed the strongest protection in the deoxyribose assay. Nutmeg improved the stability of oils which are sunflower, corn, olive and fats which are butter and margarine against oxidation at 110C. Trolox Equivalent Antioxidant Capacity (TEAC) assay is used to provide a ranking order of antioxidant activity. When used this assay, the antioxidant capacity of nutmeg was found to be higher than BHT. According to Murcia et al. (2004), they reported that phenylpropanoid compound extracts from nutmeg possessed antioxidant activity.

Recently Checker et al., (2008) observed that lignans present in aqueous extract of fresh nutmeg mace possess antioxidant, radioprotective and immunomodulatory effects in mammalian cells. High antioxidant activity has been reported in monoterpenoid rich extracts such as terpinene-4-ol, alpha-terpineol and 4- allyl-2,6-dimethoxyphenol in nutmeg seed (Maeda et al., 2008). According to Yadav and Bhatnagar (2007) , they reported that aril part of M. fragrans have significant antioxidant activity due to its ability to


inhibit lipid peroxidation and superoxide radical scavenging activity in rat. Pretreatment with M. fragrans effectively protects the mice against radiation-induced biochemical alterations as evident by decrease in lipid peroxidation level and acid phosphatase activity and simultaneous increase in hepatic glutathione and alkaline phosphatase activity (Sharma and Kumar, 2007). 2.2 Longan (Dimocarpus longan)

Figure 2.2 Seeds of Dimocarpus longan Source : Crane et al., (2005)

2.2.1 Illustrated taxanomy Kingdom Division Class Order Family Genus Synonym names : Plantae : Magnoliophyta : Magnoliopsida : Sapindales : Sapindaceae : Dimorcarpus Lour. : Nephelium longan (Lam.) Carm.; Euphoria longana Steud. Scienctific names : Dimocarpus longan Lour.

Source: Crane et al., (2005)


2.2.2 General information Longan, Dimocarpus longan Lour., the most popular members in Sapindacea family. This fruit is a close cousin with lychee. This fruit is a highly attractive subtropical fruit widely distributed in the south of China. It is a tree fruit that grows in clusters. The individual fruits are round with a diameter of about 1 inch and are covered with a brown skin that has a smooth texture. Inside the fruit is a single, round seed. It produces fruit with sweet, translucent and juicy flesh. The flesh of the longan is translucent, white and crisp. It can be eaten fresh, frozen, canned, dried, or processed into juice, wine, pickles, preserves, ice-cream and yoghurt. Longan is a small, round, undistinguished looking fruit. The brittle light brown skin encloses delicious translucent, juicy soft flesh around a single large, black inedible pit. The Chinese name for this fruit is long yan rou, which literally means dragon eye flesh. Longans are adapted to tropical and warm subtropical areas with high rainfall. They grow and crop best in areas with short cool frost-free winters and long hot humid and wet summers. The temperature regime for fruit set and development is similar to that for lychee, but, the minimum temperature required inducing panicle and flower initiation appears to be less. According to Morton (1987) longan seeds are traditional used as a folklore medicine, which are administered to counteract heavy sweating and the pulverized kernel serves as a styptic. Longan seeds have previously been shown to possess potent antioxidant activities which could be ascribed to their phenolic contents (Soong and Barlow, 2005). However, there is a little study on longan peel which usually regards as a waste material. No previous study on the antioxidant property of longan seed so far as we know. 2.2.3 Antioxidant properties of longan Longans have higher in sugar and contain several vitamins and minerals. Longan seeds have been found to be a rich source of antioxidant phenolic compounds which are promising as functional food ingredients or natural preservatives. According to Soong and Barlow, (2005) reported longan seeds


contained high levels of gallic acid, corilagin and ellagic acid. It has been proven to possess strong free radical scavenging activity (Rangkadilok et al., 2005). However, Rangkadilok et al., (2005) reported that the afore mentioned three characterised polyphenols might not be the only contributors for the high antioxidant activity of longan seeds. Instead, other phenolic constituents might also play important roles. On the other hand, besides gallic acid and ellagic acid, many other phenolic glycosides such as monogalloyl-glucose, monogalloyl-diglucose, digalloyl-diglucose, penta - to heptagalloyl-glucose, hexahydroxydiphenoyl ellagic acid-pentose conjugate, galloyl-



glucopyranose, etc., were found in longan seeds by HPLCESIMS analysis (Soong and Barlow, 2005). However, neither the complete structures of these polyphenols nor their individual antioxidant activities have been determined. 2.2.4 Food exploitation Longans can be dried. For drying, this is the oldest processing method known. It was developed in China before other technologies for preserving the fruit became available (Chen and Huang, 2001). For this processing, the fruits are first heated to shrink the flesh and facilitate peeling of the rind. Then the seeds are removed and the flesh dried over a slow fire. The dried product is black, leathery and smoky in flavor and is mainly used to prepare an infusion drunk for refreshment. A liqueur is made by macerating the longan flesh in alcohol. Longans also can be canned, juiced and frozen (Subhadrabandhu and Yapwattanaphun, 2001).




Snake fruit (Salacca edulis Reins.)

Figure 2.3 Snake fruit pulp and seeds. Source: Anon, (1998)


Illustrate taxanomy Kingdom (unranked) (unranked) (unranked) Order Family Genus Species Binomial name Synonym names : Plantae : Angiosperms : Monocots : Commelinids : Arecales : Areacaccae : Salacca : S. zalacca : Salacca zalacca : Calamus zalacca, Salacca edulis

Source: Anon, (1998)


General information This fruits have been called The Future of Our Health and The Superheroes of Functionality (Starling and Shane, 2007). Snake fruit or salak (Salacca edulis Reinw) belongs to the class of Salacca originated from South East Asia. This fruit is egglike in shape. The skin of the fruit is brown and looks like a snake skin. It contains three pieces of seeds covered with white flesh. In Indonesia there are many snake fruit cultivars; however, most of them have an astringent taste and are not sweet.


Snake fruit is a spiny palm. It is not a form of trunk but rather sprouts the leaves from the ground level. Fruits are in tight, globose bunches, and round. The fruit skin is covered with regularly arranged scales, giving the appearance of a reptile skin. The edible part is the aromatic and translucent whitish pulp, resembling in taste a mixture of pineapple and banana. Each fruit contains 1 to 3 dark brown seeds. The pulp is edible and consists of three lobes. The lobes have the consistency of three large peeled garlic cloves, yet the taste is sweet and acidic with an apple-like texture.

In Indonesia, snake fruit is widely cultivated in the lowlands throughout the islands. There are many different snake fruit cultivars; each of those has its particular taste and fruit characteristics. Regardless, the present major problems in the development of snake fruit production are quality, quantity and continuity of supply. Currently, there is an increasing interest in investigating snake fruit production techniques and postharvest properties in Indonesia.

2.3.3 Antioxidant properties of snake fruit In a study published by European Food Research and Technology (Leontowicz et al., 2006), snake fruit was found to contain a high concentration of bioactive compounds, high antioxidant potentials and to positively affect plasma lipid profiles and plasma antioxidant activity in rats fed with cholesterol-containing diets. It can be identified as chlorogenic acid, epicatechin, singly-linked proanthocyanidins that mainly existed as dimers through hexamers of catechin or epicatechin (Shui, 2004).

2.3.4 Food exploitation Snake fruit usually consumed fresh as well as made in the form of candy using sugar. Malaysia mostly consumed this fruit as in original form. This is because to avoid any nutrient damage if this fruit going under treatment. There is no information about the uses of the snake fruit seed. Usually the seed is non-edible portion of the fruit and will be thrown away after finish consuming the pulp (Leontowicz et al., 2006).



Antioxidant Antioxidants may be defined as a substance, when present at low concentrations compared to oxidizable substrates, significantly delay or inhibit oxidization of those substrates. Antioxidants, as a tradition, are divided into two groups, chain breaking as primary group and preventing as secondary group (Antolovich et al., 2002). In more detailed way, antioxidants can be grouped as inhibitors of free-radical oxidation reactions, inhibitors interrupting the propagation step of autoxidation, singlet oxygen quenchers, synergist antioxidants, reducing agents and metal chalators (Pokorny, 2007).

Figure 2.4 Basic chemical structure of antioxidants Source: Pokorny, (2007) Oxidation process occurs naturally in human body and defined as electron transfer from one atom to another. Since oxygen is the ultimate electron acceptor in the electron flow system that produces energy in the form of ATP, oxidation is an essential part of aerobic life and human metabolism. But the problem may arise when electrons flow from oxidation process become unpaired and then subsequently generates free radicals, known as Reactive Oxygen Species (ROS), such as superoxide (O2-), peroxyl (ROO), alkoxyl (RO), hydroxyl (HO) and nitric oxide (NO). Free radicals are very reactive and rapidly attack molecules in nearby cells (Pietta, 2000).




Classification of antioxidant based on mechanism of action Very common way to classify antioxidants is to divide them into two mechanistically distinct groups: primary and secondary (Dapkevicius, 2002). A similar classification of antioxidants is to divide them as chain breaking, preventive and complementary (Williams and Elliot, 1997).

Primary antioxidants delay or inhibit the initiation step and interrupt the propagation step of the radical chain reaction. Antioxidants act by transferring a hydrogen atom to the peroxy radical. The resulting radicals from the oxidised antioxidant are stabilised by resonance and are relatively unreactive and therefore are not capable of initiating or propagating the oxidative reaction.

Most of the antioxidants used in food protection are primary antioxidants. Basically they are different phenolic compounds with various ring substitutions: phenolic acids, catechins, flavonoids, anthocyanidins, lignans, tannins and coumarins. Synthetic antioxidants, like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), propyl gallate (PG) also have a phenolic structure.

The effectiveness of the phenolic antioxidant depends on the resonance stabilization of the phenoxy radical. Substitution at ortho and para position increases the reactivity and formed radicals are more stable. Bulky substituents such as the tertiary alkyl groups of BHA and BHT create steric hindrance and provide stability to the phenoxy radical, however they also lower the reaction rate with peroxy radicals.

Several antioxidants are used in combinations because of synergistic effects. For instance, because of the earlier described steric hindrance of BHA and BHT, they are often used in combination with other antioxidants such as propyl gallate and TBHQ. Another class of antioxidants is the secondary or preventive antioxidants. They include metal chelating agents, singlet oxygen quenchers, peroxide destructors and some others (Wong, 1989).



2.4.2 Free radical Oxidation metabolism is an essential process for survival of living things that can causes formation of free radicals (Pourmorad et al., 2006). Although they are unwanted metabolic by-products, they are continuously released by aerobic metabolisms (Mantle et al., 2000). Free radicals can also be produced by light energy, photochemical smog, tobacco products, polyunsaturated fats, alcohol, radiation, physical stress that leads depletion of immune system antioxidants and modification of proteins caused by gene expression changes (Pourmorad et al., 2006).

Figure 2.5 Cause and effect of free radicals Source: Pourmorad et al., (2006) Free radicals are unstable. It has a tendency of being stabilized in a way of reducing their energy level by transferring their excess electron to nearby substances. As an example, when they are formed within body, they attack nearby tissues by oxidizing membrane lipids, cellular proteins, DNA that causes complete shutdown of cellular activities such as respiration and terminates the cell. Furthermore, the interaction of oxygen free radicals with members of lipidic portion of body leads to formation of new radicals such as hydroperoxides, superoxide, lipid oxides and hydroxyl radical whose type may interact with biological systems in a citotoxic manner (BenaventeGarcia et al., 2000).