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Guide Antioxidant Measurement and Applications

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The polyphenol is oxidized to the corresponding quinone, the reduction product is bis neocuproine copper I chelate shows absorption maximum at nm. Neocuproine is 2,9-dimethyl-1,phenanthroline. Liberated protons are buffered in ammonium acetate medium. The CUPRAC reagent does not involve any radical reagent hence is insensitive to number of physical parameters like, temperature, sunlight, pH, humidity etc.

The reagent can be adsorbed on a cation-exchanger membrane to build a low cost, linear response antioxidant sensor Ozyurt et al 56, CERAC assay selectively oxidized antioxidant compounds such as trolox, quercetin, gallic acid, ascorbic acid, catechin, naringin, caffeic acid, ferulic acid, glutathione and cysteine but not citric acid and reducing sugars. This method is based on the electron transfer ET reaction between Ce IV ion and antioxidants in optimized acidic sulphate medium i. In this method, increasing amounts of antioxidant were added to a fixed amount of Ce IV. For this purpose, 1 ml of 1.

After standing for 30 min at room temperature, the fluorescent product, Ce III , exhibited strong fluorescence at nm with an excitation wavelength of nm, the fluorescence intensity being correlated to antioxidant power of the original sample. The titration curve was constructed as signal intensity against antioxidant concentration.

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Lipid peroxidation inhibition assay LPO : Lipid peroxidation, a well-established mechanism of cellular injury in plants and animals, is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides are unstable and decompose to form a complex series of compounds including reactive carbonyl compounds. One molecule of either MDA or 4-hydroxyalkenal reacts with 2 molecules of N-methylphenylindole to yield a stable chromophore carbocyanine dye with maximal absorbance at nm as indicated in Figure 6a 58, Thiobarbituric acid reactive substances TBARS assay: The measurement of thiobarbituric acid reactive substances is a well established method for screening and monitoring lipid peroxidation A standardized solution of Fe-EDTA complex reacts with hydrogen peroxide by a Fenton type reaction, leading to formation of hydroxyl radicals.

Antioxidants from the added sample human and animal tissues, fluids, drugs and food cause suppression of the production of TBARS. This reaction can be measured colorimetrically at nm or fluorometrically at an excitation wavelength of nm and emission wavelength of nm 61, The fluorescence decay curve is monitored in the absence and presence of antioxidant which is the index of the hydroxyl radical prevention capacity. Gallic acid is chosen as a reference standard and activity is measured in terms of Gallic acid equivalents GAE.

The hydroxyl radical prevention capacity is mainly due to the metal-chelating capability of the compounds Ferrous ions chelating assay: One of the mechanism of antioxidative action is chelation of transition metals preventing catalysis of hydroperoxide decomposition and Fenton type reactions 64, In the presence of chelating agents, the complex formation is disrupted leading to colour reduction.

Measurement of colour reduction allows the estimation of the chelating activity of the coexisting chelator. A volume of 1. The mixture was mixed thoroughly and incubated for 5 min. Ferrozine react with diavalent iron to form a stable red or purple complex species that is very soluble in water.

EDTA is usually added to check the stability of metal complex Nitric oxide free radical scavenging activity: Sodium nitroprusside in aqueous solution at physiological pH generates nitric oxide, which interacts with oxygen to produce nitric ions that can be estimated by use of Griess reagent 67, 68, Scavenger of nitric oxide competes with oxygen leading to reduced production of nitric oxide.

The absorbance of chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphylethylenediamine was read at nm. Colour of the solution changed from colourless to light pink to deep purple. The percentage scavenging of nitric oxide of plant extract and standard solution of potassium nitrite is calculated using the following formula:.

Substances having a reduction potential react with potassium ferricyanide forming potassium ferrocyanide which further reacts with FeCl 3 to form an intense prussian blue complex having maximum absorbance at nm. The amount of complex formed is directly proportional to the reducing power of test sample PCL Assay: In the PCL assay Photochemiluminescence the photochemical generation of free radicals is combined with the sensitive detection by using chemiluminescence 71, The free radicals are visualized with a chemiluminescent detection reagent.

Luminol 5-amino-2, 3-dihydro-1,4-phthalazinedione acts as photosensitiser and oxygen radical detection reagent. For the analysis 1. The determination is done at pH 5. The DMPD radical cation is stable upto 12 h. This assay can equally be applied to both lipophilic and hydrophilic antioxidants.

(PDF) Measurement of antioxidant activity | joy zhong - gyqacyxaja.cf

This method is rapid and inexpensive and reproducible. It has a promising aspect of use in screening large number of fruit samples Cellular Antioxidant Activity CAA : The cellular antioxidant activity model better represents the complexity of biological systems and is an important tool for screening foods, phytochemicals and dietary supplements for potential biological activity.

Many of the chemical assays are performed at non physiological pH and temperature and may therefore be unreliable indicators of true biological antioxidant levels.

The technique accounts for some aspects of uptake, metabolism and distribution of antioxidant compounds within cells, so it provides a clearer picture of how the antioxidants act within a living cell in vivo and by extension, a living cell culture rather than in a test tube in vitro. Although CAA helps in determining actual efficacy of antioxidants within the body of animal as it takes into account aspects of cellular uptake, distribution and metabolism of antioxidant compounds but the method used is very expensive and is not suitable for initial antioxidant screening of foods and dietary supplements.

Since liver is the major place for xenobiotic metabolism therefore liver cells can be used as model cells for determination of oxidative stress in cultured cells for evaluation of chemoprotective effect of dietary compounds. Human HepG2, a differentiated cell line of hepatic origin is used as reliable model for such assays The concentration of DCF, a fluorescent compound, can be measured using a fluorescent plate reader.

The assay involves the use of peroxyl radicals generated from azobis amidinopropane dihydrochloride ABAP. When a sample of phytochemical origin such as fruit or vegetable extract or dietary supplements containing antioxidants is added to the assay, the antioxidants react with the peroxyl radicals, preventing the peroxyl radicals from oxidizing the DCFH and thereby preventing the formation of DCF.

Consequently the fluorescence decreases due to the scavenging effects of the antioxidants. The representative examples of antioxidants include vitamins, carotenoids, phenolics and flavonoids It relies on differences in scattering properties between lysed and intact human erythrocytes. AAPH, a peroxyl radical generator is used to enhance lipid peroxidation. The consequent hemolysis triggered a loss of the light-scattering ability in the lysed erythrocytes. When an antioxidant is added, the area under the absorbance decay curve AUC was linearly proportional to the concentration of antioxidant compound.

This erythrocyte cellular antioxidant activity ERYCA method is found to be relatively fast, sensitive, accurate, and repeatable, even when using erythrocytes from different donors and for different storage times The ERYCA assay has the advantage of assessing different mechanisms of antioxidant protection, including direct scavenging of free radicals in the surrounding medium and cell mediated antioxidant protection Cell-MAP , in one step.

Cell-MAP addresses the following: the physiochemical properties of antioxidants such as their lipo-solubility, the ability of both lipid and water soluble compounds to diffuse effectively into lipoproteins and cell membranes and eventually enhance from there, the erythrocytes defenses through mediation of both, plasma membrane redox system PMRS , and the anti oxidative defense enzyme system Obesity is also one of the key reasons of developing diseases like coronary disease, blood pressure, cancer, diabetes etc.

Many of these diseases can be reversed through nutritional excellence. Choosing right food in correct proportion can decrease the risk of life threatening disorders and increase life longevity. The antioxidant potential measurement assays can help in choosing naturally occurring antioxidant rich food containing ascorbic acid, vitamin E, carotenoids and natural polyphenols for preservation of food and decreasing the ROS in body.

HAT and ET mechanisms occur parallel in most systems although one may dominate depending upon the antioxidant structure and properties. No single assay can be used as universally accepted method for determination of total antioxidant potential. It may be concluded that raw and mildly processed fruits, vegetables, nuts, dry fruits, cereals, spices and condiments have significantly higher level of bioactive compounds richer in antioxidants 52, Fruits like blueberry, cranberry, blackberry, prunes, raspberry, strawberry, red apples, cherry, plums, peaches, grapefruits, lemons, oranges, kiwi, pomegranate are richer in antioxidant content 80, 81, Among vegetables garlic, red, yellow and white onions, red and green peppers, white and green cabbage, leek, spinach, beetroot, broccoli have higher health benefits due to their phenolic compounds 83, 84, Tomatoes contain lycopene, a stable, active antioxidant.

Many vegetables contain quercetin and related polyphenolic compounds. Tea is a source of epigallocatechin gallate, in green tea, and the aflavin and the associated thearubigins, in black tea. Red wine contains resveratrol The higher antioxidant activities among cereals were found in buckwheat, oats, amaranth, quinoa and rice Oilseeds like flaxseeds, sesame seeds, sunflower seeds, rapeseeds have high lignin content contributing towards their antioxidant potential Chemiluminescence and fluorescence assays are also being used for determining quality parameters of edible oils, such as oxidative stability, antioxidant activity, and lipid hydroperoxides content, as well as classification or adulteration of vegetable oils.

Assessment of the antioxidant activities done by Paganga et al of a serving of g fresh weight fruit, vegetable and comparison with ml beverage in micromol Trolox equivalents showed that the antioxidant activities of 1 glass ml red wine equivalent to 12 glasses white wine equivalent to 2 cups of tea equivalent to 4 apples equivalent to 5 portions of onion equivalent to 5.

Antioxidants derived from naturally occurring plant and plant derived products have gained importance in recent years as the synthetic antioxidants like BHA, BHT and TBHQ have restricted use in foods suspected carcinogens and the quest for finding better natural antioxidants and a universal method for their determination is still on. Sunita Rattan and Head of Department, Dr. Sangeeta Tiwari of Chemistry department, Amity Institute of Applied Sciences, Amity University Uttar Pradesh; India for providing the necessary infrastructure and financial support to carry out this work.

The author would also like to express sincere gratitude to the founder president of Amity University, Dr. Chauhan, whose motivation and vision gave the inspiration for this review. Int J Pharm Sci Res ; 6 2 : Article Information Sr No: 6. Download: Cited By: Published: 01 February, Role of Antioxidants An antioxidant is a molecule capable of inhibiting the oxidation of another molecule. The principle enzymatic antioxidants are the following: Superoxide dismutase SOD : Assisted by copper, zinc, manganese and iron, SOD breaks down superoxide which plays a major role in lipid per oxidation into oxygen and hydrogen peroxide.

Water-Soluble Hydrophilic and Lipid-Soluble Lipophilic Antioxidants Another categorization of antioxidants is based on whether they are soluble in water hydrophilic or in lipids hydrophobic.

Antioxidant Extraction and Determination through DPPH assay

This can be summarized as follows: The degree of hydroxylation and the positions of the —OH groups in the B ring, in particular an ortho-dihydroxyl structure of ring B catechol group results in higher activity as it confers higher stability to the aroxyl radical by electron delocalisation or acts as the preferred binding site for trace metals. However, under some conditions, such compounds may act as pro-oxidants, thus counteracting the antioxidant effect.

A double bond between C-2 and C-3, conjugated with the 4-oxo group in ring C enhances the radical scavenging capacity of flavonoids. A double bond between C-2 and C-3, combined with a 3-OH, in ring C, also enhances the active radical scavenging capacity of flavonoids, as seen in the case of kaempferol. Substitution of the 3-OH results in increase in torsion angle and loss of coplanarity and subsequently reduced antioxidant activity.

Significance of Antioxidant Potential of Plants and its Relevance to Therapeutic Applications

Substitution of hydroxyl groups in ring B by methoxyl groups alters the redox potential, which affects the radical scavenging capacity of flavonoids Quercetin Fig. Xanthohumol a chalcone and isoxanthohumol and 6-prenylnaringenin flavanones are the major prenyl-flavonoids found in beer. Xanthohumol is a more powerful antioxidant than vitamin E or genistein but less potent than quercetin. The prenyl group plays an important role in the antioxidant activity of certain flavonoids. A flavonoid chalcone chalconaringenin and a flavanone naringenin with no prenyl groups act as pro-oxidants, i.

Genistein, an isoflavone in soy also has high antioxidant potential Antioxidant Capacity The total antioxidant capacity or antioxidant activity is a meaningless term without the context of specific reaction conditions such as temperature, pressure, reaction medium, reference points, chemical reactivity etc. The chain breaking mechanisms are represented by: L. Here, L. Various Antioxidant Capacity Assays A number of protocols have been proposed to determine the antioxidant capacity.

J Agric Food Chem. Chapter 6. Springer-Verlag Berlin Heidelberg, ; Davies, Michael J. Error rating book. Refresh and try again. Open Preview See a Problem? Details if other :. Thanks for telling us about the problem. Return to Book Page. Chi-Tand Ho. Antioxidant Measurement and Applications will provide an excellent account of lipid oxidation and oxidative stability of food lipids and the role of antioxidants in prevention of oxidation, their occurrence and sources.

Oxidation in Foods and Beverages and Antioxidant Applications

There is a need in the market for a book of this caliber and will be well received by the industry. The book will cover mechanisms of action of antioxidant Antioxidant Measurement and Applications will provide an excellent account of lipid oxidation and oxidative stability of food lipids and the role of antioxidants in prevention of oxidation, their occurrence and sources. The book will cover mechanisms of action of antioxidants and analytical procedures and furthers the potential health benefits of antioxidants and their role in disease risks.

Get A Copy. Hardcover , pages. Availability: This title is currently not available. Summary Antioxidant Measurement and Applications will provide an excellent account of lipid oxidation and oxidative stability of food lipids and the role of antioxidants in prevention of oxidation, their occurrence and sources. There is a need in the market for a book of this caliber and will be well received by the industry.

The book will cover mechanisms of action of antioxidants and analytical procedures and furthers the potential health benefits of antioxidants and their role in disease risks. All Rights Reserved.


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