Antioxidants , their properties , uses in food products and their legal implications

Oxidation decreases consumer acceptability of food by changing its organoleptic properties, destroying essential nutrients and producing toxic compounds. Antioxidants delay oxidation of lipids in foods as well in human systems. Studies reveal that synthetic antioxidants may trigger diseases in human when consumed over a certain concentration. The toxicological effects of synthetic food antioxidants have been the focus of controversy in recent years. There is scope to use natural antioxidants, present in many components of food and plant sources, as a preservative. In this review different synthetic and natural antioxidants present in various foods, reactions with food and the biological system, extraction techniques and their pitfalls as well as legal implication are discussed.


Introduction
Antioxidants are a group of substances which, when present at low concentrations, in relation to oxidizable substrates, significantly inhibit or delay oxidative processes, while often being oxidized themselves.The applications of antioxidants have been widespread in the food industry for decades; and are in use in preventing lipids from oxidative degradation (Vaya & Aviram, 1999).Free radical reactions play an important role in oxidative degradation of lipids by the production of peroxides and their derivatives, which produce off-flavors in foods (Gordon, 2001).Antioxidants protect cells against the effects of harmful free radicals.There exists scientific evidence that the excessive production of free radicals in the organism, and the imbalance between their concentration and the antioxidant defences, may be related to processes such as aging (Calabrese & Maines, 2006) and diseases such as cancer (Johnson, 2001;Mantovani et al., 2003), atherosclerosis (Siekmeier, Steffen, & Maerz, 2007), stroke (Spence, 2006), rheumatoid arthritis (Firuzi et al., 2006), neurodegeneration (Droge & Schipper, 2007), inflammatory disorders (Geronikaki & Gavalas, 2006) and diabetes (Rahimi, Nikfar, Larijani, & Abdollahi, 2005).Many studies have revealed that antioxidants may play an important role in preventing diseases caused by free radicals.With evolving health consciousness in the consumer, food for well-being and health is the new trend that has caught the attention of food processors and sci-entists equally.This has led to incorporation of antioxidants in foods from various sources.A report from market analysts revealed that consumer pressure and revised legislation is causing a shift in antioxidant trends.From the year 2000 to 2009, the € 15 million total European antioxidant market was estimated to grow by a mere 1.7 per cent; while a compound annual growth rate of 35 % was foreseen for natural antioxidants (Ibbotson, 2008).Lipid oxidation has long been recognized as a major problem in the storage of fatty foods.Oxidative changes can result in repugnant flavors, destruction of valuable nutrients and even generate toxic compounds leading to economic loss to the producer and consumer.Unsaturated fatty acids are the potential sources of oxidative offflavors in fatty products and significantly contribute to the shelf life of many products such as ghee (clarified butter), butter, fat spread, milk powders, etc. (Madhavi, 1995).The term antioxidant originally was used to refer specifically to a chemical that prevents the consumption of oxygen.According to the Prevention of Food Adulteration (PFA) Act, antioxidant means "a substance which when added to food retards or prevents oxidative deterioration of food and does not include sugars, cereal oils, flours, herbs and spices" (PFA -Prevention of Food Adulteration, 2008).Early research on the role of antioxidants in biology focused on their use in preventing the oxidation of unsaturated fats.They function as free radical acceptors, thereby terminating the oxidation process at the initial step in autoxidation (Bodnar, 2010).The major antioxidants currently used in foods are monohydroxy or polyhydroxy phenol compounds with various ring substitutions.These compounds have low activation energy to donate hydrogen.The resulting free radical does not initiate another free radical due to the stabilization of delocalized radical electron, and is not subject to rapid oxidation due to its stability; the free radicals can react with lipid free radicals to form stable complex compounds (Pokorny, 2001).

Major Oxidative Changes In Food Products
The storage of fat-containing food materials is limited by the period of slow oxidation, where the sensory value is still acceptable.The stage of very slow oxidation in the beginning of storage is called the induction period.The induction period, and thus the shelf life, may be prolonged by addition of antioxidants, which are not able to entirely eliminate the oxidation reactions even when they are active in prolonging the storage time.Several types of rancidity exist in fats, oils and fatty foods, not always connected with oxidation reactions.The most important types are listed in Table 1 (Pokorny, 2008).Fats, oils and related compounds, which turn rancid on oxidation, are mostly oxidized by oxygen in the air that penetrates foods and is dissolved in both aqueous and lipid phases.If the reaction is catalyzed by enzymes, the oxidant is still air oxygen.Other oxidants (Table 2) (Pokorny, 2008) are of minor importance.In the presence of photo-sensibilizers (such as chlorophylls) and in light, ordinary triplet oxygen is converted into singlet oxygen, which is 1500 times more reactive (Bradley & Min, 1992).Fats and oils are highly vulnerable to oxidation, which can be initiated by enzymatic and nonenzymatic mechanisms.Singlet oxygen can be generated by the photosensitization of molecular oxygen by lights which in turn initiate the lipid oxidation by reacting with unsaturated fatty acids of food products (Adegoke et al., 1998).Oxidation of β-carotene and vitamin A results in release of more polar oxidation products.The oxidative destruction of the fat soluble vitamins (A, D, E and K) has important nutritional implication.Some amino acids can also undergo oxidation along with the oxidizing lipids (Min, 2007).Food lipids are susceptible to the oxidation process, which takes place in different steps like initiation, propagation and termination (Brien & Connor, 2003).Different chemical mechanisms are responsible for the oxidation of fats and oils during processing, storage and cooking.Some of these mechanisms are discussed briefly in the following section.

Types of
Main substances

Autoxidation
Autoxidation is an autocatalytic complex chemical reaction in environment between unsaturated fatty acids or their esters and oxygen (molecular or singlet oxygen) mediated by a variety of catalysts.Autoxidation leads to the formation of several peroxide compounds, which are, relatively unstable non-volatile and odorless compounds, and their decomposition products (alcohols, aldehydes, ketones) impart oxidative offflavors in fat-rich food products (Gordon, 2001).The atmospheric triplet oxygen, having a radical character, reacts with radicals and causes autoxidation.The non-radical electrophilic singlet oxygen does not require radicals to react with; it directly reacts with the double bonds of unsaturated fats and oils with high electron densities, which is called type II photosensitized oxidation (Choe & Min, 2005).Fat oxidation is influenced by a range of parameters, like exposure to light, temperature, presence of pro-oxidant metals, presence of antioxidant compounds and the degree of unsaturated fat (Fox & McSweeney, 1998).

Photo-oxidation
Light accelerates lipid oxidation, especially in the presence of photosensitizers such as chlorophylls.Chlorophylls in singlet state become excited upon absorption of light energy in 1 pico second (Choe & Min, 2006).IJFS April 2013 Volume 2 pages 81-104

Thermal oxidation
Heating of oil produces various chemical changes including oxidation.The chemical mechanism of thermal oxidation is basically the same as the autoxidation mechanism.The rate of thermal oxidation is faster than the autoxidation, and the unstable primary oxidation products, hydroperoxides, are decomposed rapidly into secondary oxidation products such as aldehydes and ketones (Choe & Min, 2007).

Enzymatic oxidation
The oxidation of lipids can also be catalyzed enzymatically.Lipoxygenase is an iron-bound enzyme with Fe in its active center.Lipoxygenase oxidizes unsaturated fatty acids having a 1-cis, 4-cis-pentadiene system, resulting in oil deterioration, and oils containing linoleic, linolenic, and arachidonic acids are favored substrates (Hsieh & Kinsella, 1986).Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can also be oxidized by lipoxygenase (Wang, Miller, & Addis, 1991).

Classification of Antioxidants
Antioxidants are naturally present in a wide variety of raw materials, but can also be added to foods for additional protection against oxidation.The antioxidants which are added to food products can be natural or synthetic compounds depending on their availability, and preparations (Yanishlieva-Maslarova, 2001).

Limitations of natural antioxidants
Natural antioxidants are available from raw materials of variable composition (Cuppett, 2001), whereas, synthetic antioxidants are produced as pure substances of constant composition.Application of synthetic antioxidants, unlike natural antioxidants, is relatively easy requiring no substantial modifications of the recipe and process-ing conditions.The amount of natural antioxidant added to food products should be adapted according to analytical results obtained.It is also affected by the processing conditions like pasteurization (change in vitamin E), sterilization, boiling and evaporation which destroys some heat-labile vitamins and reduces the biological value of proteins (Pokorny, 2001).

Synthetic antioxidants
Synthetic antioxidant compounds are widely used in food products to inhibit progress of lipid oxidation.Addition of these to food products is however, prohibited in some countries.The antioxidant effects in milk rely primarily on endogenous compounds (Brien & Connor, 2003).Some of the more popular synthetic antioxidants used in many countries including India are Butylated Hydroxy Anisole (BHA), Ter-Butyl Hydroquinone or t-buthylhydroquinone (TBHQ), and esters of gallic acid (Yanishlieva-Maslarova, 2001).Butylated Hydroxytoluene (BHT) is very effective in animal fats, low-fat food, fish products, packaging materials, paraffin and mineral oils but is less effective in vegetable oils and may be lost during frying because of its steam volatility ( (Gordon & Kourimska, 1995).

Limitations of synthetic antioxidants
There are a number of controversies in the use of synthetic antioxidants.Since food additives are subjected to the most stringent toxicological testing procedures, only a few synthetic antioxidants have been used in foods for any length of time.
Since the toxicity of some synthetic antioxidants is not easily assessed, as a result a chemical may be considered safe by a country, tolerated in another country and forbidden in a third one.The carcinogenicity of BHA and BHT in experimental animals has been reported by Hocman (1981).Reports have shown that BHA has carcinogenic effects in non-rodents (pigs, monkeys) and causes lesion formation in the rat stomach whereas BHT has carcinogenic effects in the liver of rats and mice (Botterweck, Verhagen, Goldbohm, Kleinjans, & van

Mechanism of Action
Antioxidants slow down the oxidation rates of foods by a combination of scavenging free radicals, chelating pro-oxidative metals, quenching singlet oxygen and photo-sensitizers, and inactivating lipoxygenase (LOX).

Free radical scavenging
The effectiveness of antioxidants to scavenge free radicals in foods depends on the bond dissocia-tion energy between oxygen and a phenolic hydrogen, and reduction potential and delocalization of the antioxidant radicals (Choe & Min, 2006;Cao et al., 2007).Vitamin E or α-tocopherol is a methylated phenol required in the human diet.Phenolic compounds primarily inhibit lipid oxidation through their ability to scavenge free radicals and convert the resulting phenolic radicals into a low-energy form that does not further promote oxidation.The synergistic effect of tocopherol and ascorbic acid is shown in Figure 1.The tocopherol radical sometimes reacts with lipid peroxy radicals at very high concentration and produces tocopherol peroxide.Tocopherol peroxide produces two isomers of epoxy-8α-hydroperoxytocopherone which becomes epoxyquinones upon hydrolysis (Liebler, Baker, & Kaysen, 1990).This reaction produces alkoxy radicals, instead of peroxy radicals, and loses only tocopherol.Since there is no net decrease in free radicals in the system, tocopherol does not act as an antioxidant (Choe & Min, 2009).

Pro-oxidative metals as chelating agents
Metals reduce the activation energy of the oxidation reaction, especially in the initiation step, to accelerate oil oxidation (Jadhav, Nimbalkar, Kulkami, & Madhavi, 1996).The activation energies for the autoxidation of refined bleached and deodorized soybean, sunflower and olive oils were 17.6, 19.0 and 12.5 kcal/mol, respectively (Lee, Kim, & Choe, 2007).Metals catalyze food radical formation by abstracting hydrogen.They also produce hydroxyl radicals by catalyzing decomposition of hydrogen peroxide (Andersson, 1998) or hydroperoxides (Benjelloun, Talou, Delmas, & Gaset, 1991).Ferric ions decrease the oxidative stability of olive oil by decomposing phenolic antioxidants such as caffeic acid (Keceli & Gordon, 2002).Flavonoids are known to exhibit a strong metal chelating activity in addition to their antioxidant properties, with the arrangement of 4-keto and 5-OH, or 3' and 4'-OH substituents resulting in the formation of chelating complexes between flavonoids and divalent cations (Cheng & Breen, 2000).Kolak, Ozturk, Ozgokce, and Ulubelen (2006) reported that extract of roots of Delphinium linearilobum (Trautv.)N. contains the alkaloid lycoctonine which exhibits the highest metal chelating activity.

Quenching Singlet Oxygen
Carotenoids with nine or more conjugated double bonds are good singlet oxygen quenchers by energy transfer.The singlet oxygen quenching activity of carotenoids depends on the number of conjugated double bonds in the structure (Min & Boff, 2002;Foss et al., 2004) and the substituent in the β-ionone ring (Di Mascio & Sies, 1989).βcarotene and lycopene which have 11 conjugated double bonds are more effective singlet oxygen quenchers than lutein which has 10 conjugated double bonds (Viljanen, Sunberg, Ohshima, & Heinonen, 2002).The presence of oxo and conjugated keto-groups, or cyclopentane ring in the structure increases the singlet oxygen quenching ability (Di Mascio & Sies, 1989).β-carotene, tocopherols, ascorbic acid, amino acids, peptides and phenolics are oxidized by singlet oxygen and they are all chemical quenchers of singlet oxygen (Michaeli & Feitelson, 1994).

Addition of Antioxidative Compounds to Food
The applications of antioxidants are widespread in the food industry (Zhang, Zhu, & Wang, 2004).Many studies are focused on utilizing more effective antioxidants from natural sources (Mc-Carthy et al., 2001).Spices including cloves, cinnamon, black pepper, turmeric, ginger, garlic and onion are used widely and exhibit antioxidative activities in a variety of food systems in fresh and dried form (Ramanathan & DAS, 1993; Thorat, Mohapatra, Sutar, Kapdi, & Jagtap, 2012).

Inhibition of oxidation in meat and meat products
The shelf life of processed meat can be extended by using antioxidants and proper packaging materials.Because of the growing consensus on the potential health hazard posed by synthetic antioxidants (Jones, 1992), there is a renewed interest in the increased use of naturally occurring antioxidants.Spice and herb extracts have been reported to contain compounds with antioxidant activity, when used in food systems (Fereidoon & Champaign, 1997).
Antioxidant properties of natural plant extracts in meat were reported by Ahn, Grun, and Fernando (2002).Besides spices, extracts formed from the condensation of sugar and amino acids have been reported to possess promising antioxidant activity (Wijewickreme & Kitts, 1998;Bailey, Shin Lee, & Dupy, 1987;Chuyen, 1998;Lingnert & Eriksson, 1981).One of the naturally occurring antioxidants most widely used in the meat industry is ascorbic acid, with a varied chemistry.Ascorbic acid and its esters function as antioxidants by protecting double bonds and scavenging oxygen (St. Angelo, Vercellotti, Jacks, & Legendre, 1996;Verma & Sahoo, 2000).It has complex multifunctional effects.In cured meats, ascorbic acid can accelerate color development, inhibit nitrosamine formation, and prevent oxidation and color fading.In fresh meat it is used to prevent oxidation and color fading during storage.

Inhibition of oxidation in meat and meat products
Milk and dairy products contain several antioxidants components endogenously, i.e. tocopherols, ascorbic acid, carotenoids, thiol groups of skim milk, and some products of maillard reaction (Fox & McSweeney, 1998).The reaction of oxygen with unsaturated fatty acids is the major means of deterioration in ghee and almost all fat-rich dairy products.Several authors have shown that the addition of certain milk components prior to or after heat treatment will markedly slow down the autoxidation process in ghee and dairy products (Shantilal, 1984).Also some natural components added to dairy products like ice cream, flavored milk, etc. during or after processing have proven antioxidant activity including amla (Indian gooseberry) juice, aromatic herbs (rosemary, sage, fennel and rue), betel, curry and drumstick leaves, mango seed kernel, onion skin extract, tulsi (Indian sacred basil) leaves, sorghum grain powder, orange peel and black cumin, ragi powder, soybean seed phospholipids etc (Trivedi, 2008).

Inhibition of lipid oxidation
The relentless intrusion of oxygen and pervasive nature of initiating pro-oxidants, such as preformed peroxides, make it virtually impossible to prevent oxidation deterioration in general.The obvious corollary is that we must try to minimize the initiation of oxidation as well as retard the propagation reaction in order to extend the useful life time of affected food product.This oxidative deterioration can be inhibited by removal of reactants (O 2 present in air through packing) and certain conditions such as light (Yanishlieva-Maslarova, 2001) and metals (copper, iron and nickel) which act as pro-oxidants (Fox & Mc-Sweeney, 1998) and promote the oxidation reaction.The use of antioxidants, which are usually operative at the propagation level, will slow down or inhibit the oxidation process (Gunstone & Honorary, 2004).Extracts rich in antioxidative compounds from natural sources such as rapeseed oil byproduct extracts (Thiyam, Kuhlmann, Stockmann, & Schwarz, 2004), rice bran (Iqbal, Bhanger, & Anwar, 2005) and red grape marc extracts (Bonilla, Mayen, Merida, & Medina, 1999) have been reported to be used as endogenous antioxidants instead of commercial antioxidants to stabilize refined oils.Jayathilakan, Sharma, Radhakrishna, and Bawa (2007) reported that natural antioxidants, e.g.spices and ascorbic acid, acted as a promising alternative to synthetic antioxidants in controlling lipid oxidation and thus can enhance the stability of lipids and, in turn, products.
Vegetable oils are rather difficult to stabilize because of their high content of polyunsaturated fatty acids.Their advantage is the presence of natural antioxidants which, in edible oils, is mainly tocopherols.An addition of phenolic antioxidants usually shows limited efficacy, but the addition of synergists is helpful.Ascorbyl palmitate, phospholipids or organic polyvalent acids are useful as inhibitors in vegetable oils (Frankel, Huang, Kanner, & German, 1994).Gharby, Harhar, Guillaume, Haddad, and Charrouf (2012) discovered that phospholipids are a new and essential type of oil component participating in the excellent oxidative stability of edible argan oil, in addition to the Maillard reaction products, phenols and tocopherols.

Processing and Purification of Antioxidants
The growing interest in natural antioxidant compounds has resulted in overwhelming relevant scientific information.Numerous epidemiological studies demonstrating health promoting properties attributed to the antioxidants and phytochemicals present in the natural sources (David, Chirinos, & Pedreschi, 2008) have attracted the attention of many food manufacturers to produce healthy foods.Many components of milk and plants possessing antioxidant activities are used in their natural form, such as skim milk concentrates, ghee residues, phospholipids, butter milk solids, spices, betel and curry leaves, carotenes, etc.The preliminary processing of such food components may be drying (in the case of leaves or stems), milling of dried material or some other mechanical treatment.The content of active antioxidants in natural materials is usually rather low such that large additions would be necessary to obtain a significant improvement in stability against oxidation.Theoretically, it is possible to prepare nearly pure or entirely pure substances by liquid chromatography, repeated crystallization or other procedures, but further purification is not recommended.The resulting pure antioxidants would be very expensive in comparison with synthetic antioxidants; however, such large additions could have a negative effect on the flavor or functional properties of the product.It is often useful to prepare more concentrated materials (Schmidt & Pokorny, 2001).

Extraction and Identification of Selected Natural Antioxidants
There are various processes like solvent extraction, molecular distillation, and supercritical CO 2 extraction which are commercially used for the recovery of tocopherols, depending on the form of their natural sources.The first two processes have inherent drawbacks of thermal degradation and/or residual solvent, whereas the third IJFS April 2013 Volume 2 pages 81-104 method offers the advantage of selective recovery of a truly natural form of mixed tocopherols for vitamin E activity.A common feature of extraction methods is their suitability to only one plant material for certain phenolic acids or certain forms of phenolic acids.Although there are numerous methods for extraction of phenolic compounds from plant material, an optimized and validated method for extraction of a variety of phenolic compounds in different plant foods is lacking (Matilla & Kumpulainen, 2002).

Solvent extraction
Extraction of plant phenols has be carried out using various solvents such as methanol, ethanol, ethyl acetate, acetone and water at different concentrations (Peckic, Kovac, Alonso, & Revilla, 1998).The proanthocyanidins, which are oligomers of the catechin and epicatechin are highly soluble in ethyl acetate.The seeds of grapes have a higher content of proanthocyanidins than the rest of the grape.The yield of proanthocyanidins was 2.3 times higher using ethyl acetate and water (9:1) than the yield using acetone and water (2:3).It was concluded that ethyl acetate and water selectively extracts proanthocyanidins from grape seeds.Extractions of the grape pomace have been carried out using ethanol and water (80:20).The pomace extracts yielded quercetin, catechin, gallic acid and kaempferol compounds.From this study transcaftaric acid was found to be the most abundant compound present in the grape pomace (Foo & Lu, 1999).Cocoa bean husk powder and especially water and alcohol extracts were reported as potential antioxidants.Methanolic extracts of cottonseed exhibited antioxidant activity attributed mainly to flavonoids (Whittern, Miller, & Pratt, 1984).Results obtained by successive extractions of various raw materials with different solvents further indicated that the choice of the appropriate solvent depends on the raw material and the nature of the antioxidants it contains.Thus, in a sequential extraction of olive rape with hexane acetone and ethanol, the hexane extract, despite amounting to 9 % of the dried rape, had very few polyphenols and lower activity than either the acetone or the ethanol extract, which had similar behavior and amounted to 4 % and 7 % of the dried rape, respectively (Sheabar & Neeman, 1988).

Supercritical fluid extraction (SFE)
Extraction of antioxidants is mostly done by solvent extraction method; however supercritical CO 2 extraction techniques are also being practiced more recently in the pressure range of 200 -400 bar (Lee, Chung, & Park, 1991).Supercritical fluid extraction (SFE) is interesting for processing natural products because it produces extracts without organic residues, and the temperature of the process can be low, thus preserving thermo-sensitive compounds.SFE involves a high mass-transfer rate at relatively low temperatures, which is the major reason for the choice of this technique for extracting essential oils such as oleoresins, and other active principles where the final product quality is of great importance.Recently, the use of subcritical water for extraction has been exploited because by varying the temperature and the pressure of the water, the dielectric constant approached very closely the dielectric constants of some organic compounds as methanol, ethanol, butanone, polyaromatic hydrocarbons, phenol, benzene, and others (Reinoso, Moure, Dominguez, & Parajo, 2006).Subcritical water can produce extracts with a variety of organic substances with linear or ramified chains, showing the effectiveness of water as a solvent for hydrophobic organic composite extraction, due to the polarity of water measured by its dielectric constant (Miller, Hawthorne, Gizir, & Clifford, 1998).Leal et al. (2008)  ºC and at a pressure ranging from 200 to 400 bar.They reported that a simple batch process could be utilized to recover tocopherols of 40 % concentration from the esterified sludge initially containing 14 % tocopherols.The esterified soybean sludge was found to be 4 to 6 times more soluble in SC-CO 2 than the sterol-removed soybean sludge (Lee et al., 1991).For the extraction of Mentha spicata, Almeida, Mezzomo, and Ferreira (2012)

Microwave-assisted extraction of antioxidants
Microwave-assisted extraction (MAE) is a novel process that uses microwave energy to heat the solvents and the sample to increase the mass transfer rate of the solutes from the matrix into the solvent.The combination of solvents and heat is expected to increase the extraction yield as compared to other methods (Nuchter, Ondruschka, Hoffmann, & Tied, 2004).Because of the heat produced, microwave-assisted solvent extraction takes 10 to 30 minutes, whereas other methods can take hours or days to complete the extraction process.The amount of solvent used in microwave extraction is also considerably less than the amount used in other extraction processes (Shah, Richter, & Kingston, 2002).MAE is a relatively new extraction technique and has been successfully employed to extract tea polyphenols and tea caffeine from green tea leaves (Pan, Niu, & Liu, 2003), piperine from black pepper (Raman & Gaikar, 2002), capsaivinoids from capsicum (Williams, Raghavan, Orsat, & Dai, 2002), phenolic compounds from grape seeds (Hong, Yaylayan, Raghavan, Pare, & Belanger, 2001), puerarin from the herbal medicine radix puerariae (Guo et al., 2001), and color pigments from paprika (Kiss et al., 2000).Duvernay, Assad, Sabliov, Lima, and Xu (2005)

Ultrasonic extraction of antioxidants
High-intensity ultrasound has been used for preservation, cleaning of equipment, emulsification, accelerated drying and freezing, and most recently, for extraction of various compounds from food materials.Ultrasound-assisted extraction (UAE) is an attractive means of extraction as it is much simpler, faster & more effective when compared with other methods (e.g.supercritical fluid extraction) and has low instrumental requirements.Extraction is carried out using an ultrasonic probe system or an ultrasonic bath.Rostagno, Palma, and Barroso (2003)  In this process, parameters such as pressure, temperature and time duration can be varied according to the biology of the plant in order to obtain maximum yield.By HHPE, a single component of high purity can be easily obtained.Strong, weak, and non-polar compounds can all be extracted by using different solvents.Saving energy and safety are also advantages of HHPE.HHPE can be used as a tool for drug discovery, allowing chemical reactions to occur under more suitable conditions.HHPE was found to give higher yield of essential components when compared with ultrasonic and heat reflux extractions (Zhang et al., 2004).Kumar, Chu, Krishnaiah, and Bono (2006) studied the modified version of HHPE for extraction of antioxidants from M. citrifolia fruit.It was found that the antioxidant activity of the extracts increased with an increase in pressure.The optimum antioxidant activity was achieved using an extraction process at 58 ºC for 5 hours at 25 bar.

Legal Aspects of Antioxidants
Antioxidants should satisfy several requirements before being accepted for incorporation into food products.Different antioxidants show substan-tially different antioxidant effectiveness in different fats and oils and food systems due to their different molecular structures (Mikova, 2002).However, safety and acceptability factors should be considered prior to their introduction to the food system.Antioxidants to be used in food applications should posses certain properties having a positive influence on foods and consumers, such as solubility in fat, no harmful physiological effects, no objectionable flavour or color contribution to the fat, effective in low concentration, effective at a temperature between 25 and 30 ºC, stable to pH change, no destruction during processing, readily available and economical (Pokorny, 2001).

Legal aspects of natural antioxidants
The use of antioxidants in food products is governed by regulatory laws of the individual country or by internal standards.Even though many natural and synthetic compounds have antioxidant properties, only a few of them have been accepted as generally recognized as safe (GRAS) substances for use in food products by international bodies such as JECFA and the European Community's Scientific Committee for Food (SCF).Antioxidants are extensively tested for the absence of carcinogenicity and other toxic effects in themselves, in their oxidized forms and in their reaction products with food constituents.Toxicological studies are crucial in determining the safety of an antioxidant and also in determining the ADI levels.

Legal aspects of synthetic antioxidants
ADI levels for widely used antioxidants such as BHA and gallates have changed over the years mainly because of their toxicological effects in various species (Pokorny, 2001).Generally, addition of antioxidants (synthetic) is not allowed in dairy products and therefore, their stability must rely on components present in the milk.Alimentations, 2008).The results of scientific studies are controversial with links suggested between synthetic antioxidants and cancer.Scientific studies that have been carried out on rats have required very high levels of BHA to trigger cancer levels that are not relevant to normal human consumption.The type of cancer found in rats and mice was also not relevant to any digestion mechanism in humans.So scientists conclude that these findings are not relevant to humans (Food Safety, 2008).
The best method of application of natural antioxidants is to use natural food components (e.g.cereals, nuts, fruits and vegetables) because they are regarded as safe and no special approval for their application is necessary.Another possibility is to use natural food ingredients such as spices (Pokorny, 2001).Natural antioxidants are available in most of the multivitamins and there are no adverse reports or deaths through using multivitamins.However, 10 to 15 cases of vitamin A toxic reactions are reported per year in the United States, usually at doses > 100,000 IU/day for months or years (about 40 times the recommended dietary allowance) (Allen & Haskell, 2002;Meyers, Maloley, & Weeks, 1996).No adverse effects have been reported for β-carotene.Ascorbic acid toxic reactions are rare at dosages < 4 g/d.Selenium was associated with a 9 % lower risk of death.
Selenium is toxic if its consumption is more than 6 mg/ day.So far, there are no reports of adverse effects of other antioxidants; however, the safety of antioxidant use is not well documented (Vijaya & Vidyasagar, 2009).Natural antioxidants from herbs and spices occupy a special position in foods as traditional food ingredients and hence are appropriately used directly for their antioxidant characteristics.If they are applied to foods, they do not need to be declared as antioxidants (PFA -Prevention of Food Adulteration, 2008).
The safety of natural antioxidants should not to be taken for granted.Caution must be exercised, since numerous natural products are potential carcinogens, mutagens or teratogens and the safety of many natural compounds with antioxidant activity has not yet been established.A case in point is NDGA which was used extensively as an antioxidant earlier this century.
NDGA is a natural constituent of the creosote bush, which was removed from GRAS status when unfavorable toxicological results were reported (Reishe & Eitemmiller, 2008).In India, as well as in European legislation, there are no legal rules regarding declaration of natural antioxidants present in food, the permissible safe limit for consumption and at what concentration they will act as pro-oxidants and synergists; there is also a need to determine daily intake doses which should be declared on the product labels.

Conclusions
Antioxidants play a major role in preventing oxidation of lipids in foods as well in humans.Synthetic antioxidants are involved in the triggering of diseases in humans at a certain concentration.Many components of milk and plants have antioxidative properties which can be manipulated for use in foods.The toxicological effects of food antioxidants have been the focus of controversy in recent years.Better understanding of the role of natural antioxidants on food stability and human health is required.There are no safety regulatory controls over natural antioxidants at higher concentrations as they may also cause problems to human health.Toxicological studies are mainly carried out to establish the no-effect level for an ADI for humans.

IJFS April 2013 Volume 2 pages 81-104
The laws must be modified to ensure the integration of these issues in determining the criteria for safety.It must be known about the long term consequences of more doses of antioxidants.
The regulatory and judicial system must be prepared to accept this expanded role for food in the matrix of national life and the international market.
palmitate or ascorbyl strearate (or the sum of both) 0-1.25 *ADI, acceptable daily intake; bw, body weight; E number refers to food additives.

Table 4 :
Antioxidant compounds in selected vegetables and their products

Table 5
According to Codex Alimentarius, the antioxidants covered by the standard and their maximum levels of use are based in part on the food additive provisions of previously established Codex Commodity Standards, or upon the request of governments after subjecting the requested maximum levels to an appropriate method which would verify the compatibility of a proposed maximum level with the ADI (Codex