Synopsis

Evaluation of Radicals Scavenging Activity of Peristrophe paniculata Free Radicals

Radicals (often referred to as free radicals) are atoms, molecules, or ions with unpaired electrons or an open shell configuration. Free radicals may have positive, negative, or zero charge. With some exceptions, the unpaired electrons cause radicals to be highly chemically reactive. Free radicals play an important role in combustion, atmospheric chemistry, polymerization, plasma chemistry, biochemistry, and many other chemical processes. A free radical is any chemical species capable of independent existence possessing one or more unpaired electrons. Biological free radicals are thus highly unstable molecules that have electrons available to react with various organic substrates.

Sources of free radical

Internal sources External source Physiological Factors Internal sources: These can be enzymatic reactions, which serve as a source of free radicals. These include those reactions involved in the respiratory chain, in phagocytosis, in prostaglandin synthesis and in the cytochrome P450 system. Some internal sources of generation of free radicals are mitochondria, xanthine oxidase, phagocytes, reactions involving iron and other transition metals, peroxisomes, Arachidonate pathways, exercise, ischaemia / reperfusion, inflammation. External sources: These include non-enzymatic reactions of the oxygen with organic compounds. Some external sources of free radicals are cigarette smoke, environmental pollutant, radiations, ultraviolet light, ozone, certain drugs, pesticides, anesthetics and industrial solvents. Physiological Factors Mental status like stress, emotion etc. and disease conditions are also responsible for the formation of free radicals. Oxidative Stress Oxidative Stress (OS) is a general term used to describe the steady state level of oxidative damage in a cell, tissue, or organ, caused by the Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) Reactive Oxygen Species (ROS) is a term collectively describing radicals and other nonradical reactive oxygen derivatives. These intermediates may participate in reactions giving rise to free radicals or that are damaging to organic substrates. ROS in living organisms include the following [1 A].

Radical Hydroxyl Superoxide Nitric Oxide Thyl Peroxyl Lipid peroxyl

OH O2 NO RS RO2 LOO

Non-Radical Peroxynitrite Hypochloric acid Hydrogen Peroxide Singlet Oxygen Ozone Lipid peroxide

ONOO HOCl H2O2

1g (1O2)

O3 LOOH

Reactive Nitrogen Species (RNS) are radical nitrogen-based molecules that can act to
facilitate nitrosylation Reactive Nitrogen Species (RNS) include: Nitrous oxide Peroxynitrite Peroxynitrous acid Nitroxyl anion Nitryl chloride NO OONO ONOOH NO Nitrosyl cation Nitrogen dioxide Dinitrogen trioxide Nitrous acid NO2Cl NO+ NO2 N2O3 HNO2

Diseases Involving Excessive ROS Levels

The toxic effects of O2 itself, such as the oxidation of lipids and proteins, generation of mutations in the DNA, and destruction of cell membranes. Cardiovascular diseases. Atherosclerosis. Various types of cancer. Diabetes. Neurodegenerative diseases, including Parkinsons disease and Alzheimers disease. Toxicity of heavy metals (e.g., iron). Radiation injury. Vitamin deficiency. Toxicity of certain medications.

Antioxidants: The term antioxidant originally was used to refer specifically to a chemical that prevented the consumption of oxygen. In the late 19th and early 20th century, extensive study was devoted to the uses of antioxidants in important industrial processes, such as the prevention of metal corrosion, the vulcanization of rubber, and the polymerization of fuels in the fouling of internal combustion engines [1 A ]. An antioxidant is a molecule capable of inhibiting or preventing or slowing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols. [2].

Although oxidation reactions are crucial for life, they can also be damaging; hence, plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Low levels of antioxidants, or inhibition of the antioxidant enzymes, causes oxidative stress and may damage or kill cells. As oxidative stress might be an important part of many human diseases, the use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. However, it is unknown whether oxidative stress is the cause or the consequence of disease. Antioxidants are also widely used as ingredients in dietary supplements in the hope of maintaining health and preventing diseases such as cancer and coronary heart disease. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials did not detect any benefit and suggested instead that excess supplementation may be harmful3. Antioxidants are classified into two broad divisions, depending on whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants react with oxidants in the cell cytosol and the blood plasma, while lipid-soluble antioxidants protect cell membranes from lipid peroxidation1. These compounds may be synthesized in the body or obtained from the diet [3]. The different antioxidants are present at a wide range of concentrations in body fluids and tissues, with some such as glutathione or ubiquinone mostly present within cells, while others such as uric acid are more evenly distributed. Some antioxidants are only found in a few organisms and these compounds can be important in pathogens and can be virulence factors5. In general, antioxidant systems either prevent these reactive species from being formed, or remove them before they can damage vital components of the cell6. However, since reactive oxygen species do have useful functions in cells, such as redox signaling, the function of antioxidant systems is not to remove oxidants entirely, but instead to keep them at an optimum level [4]. Antioxidants are absolutely critical for maintaining optimal cellular and systemic health and well-being. Naturally there is a dynamic balance between the amount of free radicals produced in the body and antioxidants to scavenge or quench them to protect the body against deleterious effects. The amount of antioxidant principles present under normal physiological conditions may be insufficient to neutralize free radicals generated. Therefore, it is obvious to enrich our diet with antioxidants to protect against harmful diseases. Hence there has been an increased interest in the food industry and in preventive medicine in the development of Natural antioxidants from plant materials. That is why plants with antioxidant properties are becoming more and more popular all over the world. Considering the importance of this area, we have listed some important in-vivo and in-vitro models for evaluating antioxidant activity [5]. Medicinal Plant Introduction Medicinal plants are various plants used in herbalism and thought by some to have medicinal properties. Few plants or their phytochemical constituents have been proven to have medicinal effects by rigorous science or have been approved by regulatory agencies such as the United States Food and Drug Administration or European Food Safety Authority. The use of plants whether herbs, shrubs or trees in parts or in whole in the treatment and management of diseases and disorders date back to pre historic days. Plant extract have

been used in folk medicine practices for the treatment of various ailments since antiquity. The medicinal properties of various plant materials and extracts have been recognized since the beginning of the 5th century. Over the past few decades, the extensive research has focused on the health effects of phytochemicals and plant derived extracts.Natural phytochemicals present at low levels in fruits, vegetables, herbs and spices offer many health benefits, but these compounds may not be effective or safe when consumed at higher dose. Plants such as herbs have been used in folk medicine for centuries in most of the cultures throughout the world. Peristrophe paniculata is one of the traditional folk medicinal plants that have been used for hundreds of years for treating diabetes, high blood pressure, cancer, eye problems and many other illnesses [6]. Generation of free radicals or reactive oxygen species (ROS) during metabolism and other activities beyond the antioxidant capacity of a biological system gives rise to oxidative stress [7]. Oxidative stress plays a role in heart diseases, neurodegenerative diseases, cancer and in the aging process [8]. This concept is supported by increasing evidence that oxidative damage plays a role in the development of chronic, age-related degenerative diseases, and that dietary antioxidants oppose this and lower risk of disease [9]. Antioxidants are the substance that when present in low concentrations compared to those of an oxidisable substrate significantly delays or prevents oxidation of that substance [10]. Apart from their role of health benefactors, antioxidants are added in foods to prevent or delay oxidation of food, initiated by free radicals formed during their exposure to environmental factors such as air, light and temperature [11]. At present most of the antioxidants are manufactured synthetically. They belong to the class of synthetic antioxidants. The main disadvantage with the synthetic antioxidants is the side effects when taken in vivo [12]. Strict governmental rules regarding the safety of the food has necessitated the search for alternatives as food preservatives [13].Plants are the potential source of natural antioxidants. Natural antioxidants or phytochemical antioxidants are the secondary metabolites of plants [14]. Carotenoids,flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols, tocotrienols etc., are some of the antioxidants produced by the plant for their sustenance. Beta-carotene, ascorbic acid and alpha tocopherol are the widely used antioxidants [15]. The objectives of the present study were to determine the antioxidant activity, total phenolic content and total flavonoid content of the extracts from Paristrophe Paniculata. fruit by ultrasonic extraction and high pressure extraction (HPE) with ethanol and ethyl acetate as solvents followed by drying in vacuum oven and spray dryer. Two methods namely peroxide value method and DPPH radical scavenging method were used to find and correlate the antioxidant activity of the extracts. In the peroxide value method the extracts were added to the peanut oil stored in an oven at 60 + 0.5 C and oxidative deterioration (formation of peroxides) of the oil was measured for 16 days. Butylated Hydroxy Toluene (BHT) was used as reference. In DPPH radical scavenging method the free radical 2, 2diphenyl-1-picrylhydrazyl (DPPH) was used to find the antioxidant (scavenging) activity of various concentrations of the extracts. Tannic acid as natural antioxidant and BHT as synthetic antioxidant were used for comparison. The total phenolic content of the extracts was estimated by Folin-Ciocalteau test and total flavonoid content by Dowd method respectively. Many factors can cause the body to produce more reactive species than are needed. These include smoking, drinking, alcohol, too much fat in the diet, too much sun exposure, too many pollutants in the air and even too much exercise.

Types of Antioxidants There are two types of antioxidants (01) Natural antioxidants (02) Synthetic antioxidants Naturally occurring antioxidants include Vitamin C (ascorbic acid) in citrus fruits, green peppers, broccoli, green leafy vegetables, strawberries, raw cabbage and potatoes vitamin E in wheat germ, nuts, seeds, whole grains, green leafy vegetables, vegetable oils like canola and Soya bean (the antioxidants present in vitamin E are called tocopherols) -carotene in carrots, squash, broccoli, sweet potatoes, tomatoes, kale, cantaloupe melon, peaches and apricots Selenium in fish, shellfish, red meat, eggs, grains, chicken and garlic. Major synthetic antioxidants in food Examples include 2- and 3-tert-butyl-4-hydroxyanisole (BHA), 3, 5-di-tert-butyl-4hydroxytoluene (BHT), propyl gallate (PG), 2, 4, 5-trihydroxybutyrophenone (THBP) and tert-butylhydroquinone (TBHQ). Effects of Antioxidants Aging and a variety of age-related conditions may be linked to oxidation processes resulting from an excess of reactive molecules. Many compounds in food have antioxidant properties by interacting with the reactive molecules. Antioxidants from food include both vitamins and minerals such as Vitamin C, E and beta-carotene, also some element such as selenium and other compounds found in plant foods such as polyphenols and flavonoids. If ROS or RNS outnumber the antioxidant stores in the body, they can inactivate enzymes oxidize lipid and damage genetic materials (DNA). These processes have been linked to aging and a variety of age-related conditions, including heart disease and cancer. Benefits of antioxidants Antioxidants are very successful when used appropriately. They should not be looked upon as curative in themselves, but can form an extremely valuable part of integrated therapy for a wide variety of conditions, ranging from allergic dermatitis to cancer prevention. Oxidants or reactive oxygen species are produced in our body during aerobic metabolism leading to many diseases such as eyes ,lungs ,brain, arthritis, cancer, ageing, cardiovascular diseases, Alzheimers disease etc. Antioxidants are the chemicals that neutralize these oxidants and prevent us from these diseases. Antioxidants also protect the skin from harmful sunrays they are edible sunscreen. [16]. Harmful effects of antioxidants Synthetic antioxidants to be less safe because they are not naturally occurring in food. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary hydroquinone (TBHQ) are commonly employed as preservatives by pharmaceutical, cosmetic, and food companies. However, they are also suspected of being responsible for liver damage and carcinogenesis[17] and toxicity [18],[19]. Therefore, the use of synthetic antioxidants is doubted. In recent years researchers are trying to replace synthetic antioxidants with natural Antioxidants such as flavonoids, tocopherols and ascorbic acid Relatively strong reducing acids can have antinutrienteffects by binding to dietary minerals such as iron and zinc in the gastrointestinal tract and preventing them from being absorbed. [20]. Notable examples are oxalic acid, tanninsand phytic acid, which are high in plant-based diets. [21].Calcium and iron deficiencies are not uncommon in diets

indeveloping countries where less meat is eaten and there is high consumption of phytic acid from beans and unleavened whole grain bread[22]. Nonpolar antioxidants such as eugenola major component of oil of cloveshave toxicity limits that can be exceeded with the misuse of undiluted essential oils [23]. Toxicity associated with high doses of water-soluble antioxidants such as ascorbic acid are less of a concern, as these compounds can be excreted rapidly in urine[24]. More seriously, very high doses of some antioxidants may have harmful long-term effects. The beta-Carotene and Retinol Efficacy Trial (CARET) study of lung cancer patients found that smokers given supplements containing beta-carotene and vitamin A had increased rates of lung cancer [25]. Subsequent studies confirmed these adverse effects. [26]. [26A]. [26B]. Antioxident level Dragland S and colleagues showed in their study entitled "Several Culinary and Medicinal Herbs are Important Sources of Dietary Antioxidants", and published in the Journal of Nutrition (2003 May) that the antioxidant level of herbs can be as high as 465 mmol per 100 g. Sources o f natural phenolic antioxidants Dietary antioxidants such as a scorbates, tocopherols and carotenoid s are well known and there is a surplus of publications related to their role in health. Plant phenols have not been completely studied because of the complexity of their chemical nature and the extended occurrence in plant materials. Extensively studied sources of natural antioxidants are fruits and vegetables, seeds, cereals, berries, wine, tea, onion bulbs, olive oil and aromatic plants. Attempts are also made to identify and evaluate antioxidants in agricultural by-products, ethnic and traditional products, herbal teas, cold pressed seed oils, exudates resins, hydrolysis products, not evaluated fruit s and edible leaves and other raw material s rich in antioxidant phenols t hat have nutritional importance and/or the potential for applications in the promotion of health and prevent ion against damages caused by radicals [27]. Experimental Work: Preparation of Samples A finely ground amount (100gms) of the fruit and seeds, or leaves or stems or roots of Pristophe paniculata were soaked in methanol in 5:250 (w/v) in cork-fitted flasks for 24 hours at 30oC and 240 rpm. The next day, extract obtained was filtered and stored at 4 oC, while the residue was re-soaked in methanol in the same proportion at 240rpm and 30oC for 24 hours. The filtrate obtained the second day was mixed with the filtrate of first day. The methanolic residue was obtained by evaporating methanol at 30oC using rotary evaporator.

Flow Sheet of Extraction

Dried Plant / Herb
Extracted with Methanol and evaporated

Crude Extract or Residue

Extracted with n-hexane

n-hexane layer was separated and evaporated

Aqueous Layer

Extracted with Ethyl acetate

Ethyl acetate layer was separated and evaporated

Aqueous Layer

Antioxidant capacity assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). [27A].

Methods for Evaluation of Radicals Scavenging Activity of Peristrophe paniculata

ABTS+ Assay ABTS+ Assay protocol, developed by Re et al. (1999) [28] was followed. 7mM ABTS solution was prepared by dissolving ABTS in doubly distilled water. ABTS radical cation (ABTS+) was developed by reacting ABTS stock solution (7mM) with potassium persulfate (2.45m M) and allowing the mixture to stand in the dark at room temperature for 12-16 hrs before use. For the study of antioxidant activity of standard antioxidant and seed samples, the ABTS stock solution was diluted either with PBS buffer (pH 7.4) or methanol to an absorbance of 0.70 (+0.02) at 734 and 745 respectively and equilibrated at 30oC. For seed extracts, the dilution was made in the respective solvents. After addition of 10l of neat or diluted sample solutions to 2.99ml of diluted ABTS+ solution (A= 0.700 + 0.020), the absorbance reading was taken at 25C exactly 1 min after initial mixing up to 8 minutes. Appropriate solvent blanks were run in each assay. All evaluations were carried out in triplicate at each separate concentration level of the standards. The percentage inhibition of absorbance was calculated by the following formula and was plotted as a function of concentration of antioxidants and of Trolox for the standard reference data. % inhibition (at 734 nm) = (1- Af /Ao) x 100 Where Ao is the absorbance of radical cation solution before addition of sample/standard antioxidants while Af is the absorbance after addition of the sample/standard antioxidants.

DPPH radical scavenging activity method (Qualitative analysis) The DPPH free radical method is based on the determination of the concentration of 2, 2diphenyl-1-picrylhydrazyl (DPPH) at steady state in a methanol solution, after adding the mixture of antioxidants. DPPH absorbs at 515 nm, and as its concentration is reduced by the existence of an antioxidant, the absorption gradually disappears with time. A Perkin Elmer UVVIS lambda 25 spectrophotometer was used and the quantity of the mixture of antioxidants needed to reduce by 50 % the initial DPPH concentration was evaluated [13]. This characteristic parameter is called efficient concentration (EC50) or oxidation index. The lower the EC50, higher is the antioxidant activity of the examined compound. The DPPH radical- scavenging activity in terms of percentage was calculated according to the following equation [14]. DPPH scavenging activity (%) = {1- (Abs515 sample / Abs515 DPPH solution)} 100% Advantages This technique is easy, effective, and rapid way to study plant extract profiles. No sample separation is needed. Potency of sample can be known. Total Phenolic Content The TPC of the extracts was determined using the Folin Ciocalteu reagent [29]. Stock solution of gallic acid was made by dissolving 0.5 g gallic acid in 10 mL of ethanol in a 100 mL volumetric flask and diluting to volume with double distilled water. 20% Sodium carbonate solution was prepared by dissolving 20 g of anhydrous sodium carbonate in 80mL of double distilled water. After boiling and subsequent cooling of the solution, a few crystals of sodium carbonate were added. The solution was allowed to stand for 24 hours, filtered and the volume was raised to 100 mL with double distilled water. To prepare a calibration curve, 0, 1, 2, 3, 5 and 10 mL of gallic stock solution were added into 100 mL volumetric flask separately and then diluted to volume with double distilled water. The resultant solutions contained concentrations of 0, 50, 100, 150, 250 and 500 mg/L gallic acid. An aliquot 40 l were taken into separate cuvettes from each standard solution and sample or blank, and to each 3.16 mL of double distilled water and 200 l Folin Ciocalteus reagent was added and mixed well. After 8 minutes, 600 L of sodium carbonate solution was mixed thoroughly in the solution. The solution was incubated at 40 oC for 30 min and noted the absorbance at 765 nm against the blank (without phenolic solution). A concentration versus absorbance linear plot was thus obtained. The concentration of total phenolic compounds of each plant extract in milligram of gallic acid equivalent (GAE) was determined by using the following standard equation. Absorbance = 0.004x + 0.0723 (Gallic acid (mg/L) Total flavonoid content Total flavonoid content was determined using a colorimetric method developed by Dewanto et al [30]. An aliquot of 250L of seeds extract or quercetin standard solutions was mixed with 1.25mL of distilled water in a test tube followed by addition of 75 L of 5% NaNO2 solution. After 6 min, 150 L of 10% AlCl3 .6H2O solution was added and allowed to stand for 5 min. Added 500 L of 1M NaOH and the volume of the mixture was raised to 2.5 mL with distilled water and mixed well. The absorbance was measured immediately against the blank at 510 nm to record the calibration curve and the correlation coefficient (R2), slope and intercept of the regression equation obtained were calculated by the method of least square. The amount of total flavonoids in the canola was expressed as mean (milligrams of quercetein equivalents per gram of extract) SD for five replications, by using the equation

obtained from the calibration curve of the antioxidant. Metal Chelating Activity The metal chelating activity of samples was estimated based on the decrease in the maximal absorbance of the iron (Fe2+)-ferrozine complex according to previously reported methods (Dinis et al., 1994) [31]. 100 L of a sample solution was added to 50 L of Ferrous Sulphate (2mM). The reaction was initiated by the addition of 200 L Ferrozine (5.0 mM), and then the total reaction volume was adjusted to 4 ml with ethanol. After the mixture had reached equilibrium (10 min), the absorbance at 562 nm was read. The control was prepared without the test compound. Fe2+ chelating activity of the test compound was calculated from the following formula Chelating activity (%) = [(Acontrol Asample)/Asample] 100 Superoxide Anion Radical Scavenging Activity Superoxide anion radical scavenging activity was determined follow ing the method previously used by N ikishimi et al. ((1972). The PMS-NADH system was used for generation of superoxide radicals. The reaction mixture contained 100 L extract, 200 M NBT, 624 M NADH, and 80 M PMS in 0.1 M phosphate buffer (pH 7.4). After 2 minutes of incubation, absorbance was measured spectrophotometrically at 560 nm. The scavenging effect was calculated using the following formula: Percent scavenging=[1 As/Ab 100] Where As and Ab are the absorbances of sample and blank solutions at 560 nm, respectively. FRAP (Ferric Reducing Antioxidant Power) 1. The FRAP assay developed by Benzie and Strain [32] was used for the quantification of water- and fat-soluble antioxidants [15-16]. FRAP reagent was prepared by mixing acetate buffer of pH 3.6 (25mL), prepared freshly 2.5 mL of TPTZ solution (10mm/L) in 40 mm HCl solution and 2.5 mL of FeCl3.6H2O (20Mm) and incubated at 370C Through out the monitoring period .Reagent blank reading was taken at 593 nm.300 ul of the freshly prepared FRAP reagent was taken and into this 10 ul of seed sample/standard antioxidant was then added along with 30ul of the double distilled water. The absorbance was noted for 4 min at 593 nm. Absorption changes appeared when the TPTZ-Fe3+ complex reduces to the TPTZ-Fe2+ form in the presence of antioxidants. An aqueous solution of 500 mol/L FeSO4 7 H2O was used for calibration of the instrument. Advantages The FRAP assay is inexpensive, reagents are simple to prepare, results are highly reproducible, and the procedure is straightforward and speedy. The FRAP assay is the only assay that directly measures antioxidants or reductants in a sample the other assays are more indirect [33]. Disadvantages FRAP cannot detect species that act by radical quenching (H transfer), particularly SH group containing antioxidants like thiols, such as glutathione and proteins. [34], [35]. The objective of the present study was to evaluate the antioxidant potential and free radical scavenging activity of Paristrophe paniculata. The extract was examined for different reactive oxygen species (ROS) scavenging activities including hydroxyl, superoxide, nitric oxide, hydrogen peroxide, peroxynitrite, singlet oxygen and hypochlorous acid, and for phenol and flavonoid contents, iron chelating capacity and total antioxidant activity.

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