Spectrophotometric Quantitation of Antioxidant Capacity through the Formation of a Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E

Abstract

A spectrophotometric method has been developed for the quantitative determination of antioxidant capacity. The assay is based on the reduction of Mo(VI)to Mo(V) by the sample analyte and the subsequent formation of a green phosphate/Mo(V) complex atacidic pH. The method has been optimized and characterized with respect to linearity interval, repetitivity and reproducibility, and molar absorption coefficients for the quantitation of several antioxidants, including vitamin E. The phosphomolybdenum method, in combination with hexane monophasic extraction, has also been adapted for the specific determination of vitamin E in seeds. The results obtained with the proposed method were validated by comparison with a standard HPLC method. The phosphomolybdenum method is routinely applied in our laboratory to evaluate the total antioxidant capacity of plant extracts and to determine vitamin E in a variety of grains and seeds, including corn and soybean.There is an increasing interest in the use and measurement of antioxidants in food, pharmaceutical, and cosmetic industries. This interest is rooted in the cumulative evidence that connects oxidative stress with numerous d egenerative d isorders ranging f rom p remature aging, pr ostaglandin-mediated i nflammatory processes, cancer, and a l ong series of diseases i n which free r adicals ar e implicated (1, 2). In addition, many states implement very rigorous regulations on the use of food preservatives, so that they only allow the use of natural antioxidants. Basically, these include vitamin C (ascorbic acid), vitamin E (tocopherols and tocotrienols), and relatively complex extracts from a number of plant species (Rosmarinus officinalis, Nerium oleander, and Myrtus comunis).The e arly works by Ch ipault et a l. (3-5) w ere t he precursors o f m any s tudies o n t he antioxidant capacity of a number of plant extracts with potential applications as preservatives in the food, cosmetics, and p harmaceutical industries (6 -12). The c urrent i nterest of our l aboratory i n t he b iosynthesis o f tocopherols in plants and in the identification of alternative sources of natural antioxidants prompted us to develop a m ethod for t he e valuation o f water-soluble a nd fa t-soluble a ntioxidant capacity. T he phosphomolybdenum method that we propose in this article is now systematically used by us in extensive screenings o f samples o f v ery different origins a nd composition i n our search f or natural sources of vitamin E and other powerful antioxidants. MATERIALS AND METHODSChemicals an d r eagents. Ammonium mo lybdate; ascorbic ac id; r educed g lutathione; b utyl hydroxytoluene (BHT);3 α-, γ-, an d δ-tocopherol; a nd t he internal standard α-tocopherol a cetate were obtained from Sigma (St. Louis, MO). HPLC gr ade methanol, h exane, ethanol, dimethyl sulfoxide, and analytical grade sodium phosphate and sulfuric a cid were from Merck (Darmstadt, Germany). All other chemicals were of analytical grade.Instrumentation. The m olecular a bsorption s pectra and absorbance a t s pecifi...

Physicochemical characterization and antioxidant activity of Palestinian honey samples

“…The TAC was estimated by the phosphomolybdenum method according to the reported procedure (Prieto, Pineda, & Aguilar, 1999). The assay is based on the reduction of Mo (VI)–Mo (V) by the honey solutions and subsequent formation of a green phosphate/Mo (V) complex in acid medium.…”

Bioactive potential of the wild mushroom Astraeus hygrometricus in South-west India

“…The TAA was assessed according to the method by Prieto et al (1999). Mushroom extract (0.1 ml) was mixed with a reagent mixture (H 2 SO 4 , 0.6 M + Na 2 SO 4 , 28 mM + NH 4 Mo 7 O 24 .2H 2 O, 4 mM) (1 ml), incubated (95°C) (90 min), cooled and absorbance of phosphomolybdenum complex was measured (695 nm) with methanol blank using ascorbic acid as standard to express TAA in μM equivalent to ascorbic acid per gram of flour (μM AAEs g −1 ).…”

In Vitro Antioxidant and Free Radical Scavenging Activity of Nardostachys jatamansi DC.

“…The antioxidant capacity of NJE was evaluated by the method of Prieto and colleagues [18]. A 0.1 ml aliquot of sample solution (equivalent to 100 mg) was combined with 1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate).…”

“…The phosphomolybdenum method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compounds and formation of green Mo(V) complex with a maximal absorption at 695 nm [18].The antioxidant capacity of NJE was estimated to be equivalent to 109.48 AE 11.72 mg of ascorbic acid/g of the dried extract.…”