Use of test systems for assessing the total antioxidant activity of seeds

Abstract

Introduction.The method using the free radical DPPH • is one of the most successful, widespread indirect methods for assessing the total antioxidant activity (TAOA). DPPH • is sufficiently selective and reacts neither with flavonoids that do not contain hydroxyl groups in the B-ring nor with aromatic acids containing only one hydroxyl group. The method is based on the ability of the stable free radical of 2,2-diphenyl-1-picrylhydrazyl (DPPH •) to react with proton donors, including phenols.

The aim and tasks of the study. The main objective of this study was to pilott the DPPH test systems for assessing the TAOA of seeds in routine analysis (screening of the plant genetic pool of Ukraine and of breeding material).

Results and discussion. The antiradical activity is determined using the stable radical (DPPH •) as described in [Arabshahi, Urooj, 2007] with minor changes: methanol in elution solution was replaced with non-toxic ethanol; the DPPH concentration was chosen so that the maximum extinction of solutions would not exceed 1.4 units. The light transmission of all solutions was within the linear part of the calibration curve. The reaction time was increased from 30 minutes to 2 hours to reduce the effect of temperature fluctuations in the laboratory (within +13°C - + 30°C) and to increase the number of samples analyzed in one experiment to 32 (in 3 replicas). Having carried out numerous experiments, we adopted the following procedure: seeds were additionally dried in a thermostat at 25°C for 4-5 days; samples were ground in a laboratory mill for 1 minute (3 × 20 sec); weighted amounts of flour (0.5 g each) were placed in vials (with sealing caps); 4.5 ml of 80% ethanol (diluted with distilled water) was added; extraction lastedat room temperature in the dark for 18 to 20 hours; samples were centrifuged (10 min at 3,000 × g) in an OPN-3 centrifuge; 2 ml of supernatants were transferred to clean vials. Ethanol solution of the radical is prepared as follows: dissolve 22 mg of DPPH in 400 ml of 80% ethanol on a magnetic stirrer under diffuse light, small grains of undissolved dye are triturated additionally with a porcelain pestle. The solution is filtered and stored during the day of analysis. Before starting the analysis, the optical density of solution obtained by adding 0.5 ml of 80% ethanol to 3.5 ml of DPPH solution is measured on a spectrophotometer at 517 nm, and if the extinction exceeds 1.4, the solution is further diluted with 80% ethanol so that its extinction will be 1.4 (working solution of DPPH • is approximately 125 μM). 0.5 ml of a seed extract is added to 3.5 ml of working solution of DPPH • at room temperature, mixed rapidly, let  to sit for 2 hours in a dark place, and then a change in light absorption of the mixture is recorded. In the control, 3.5 ml of working solution of DPPH is added to 0.5 ml of 80% ethanol. The ability of a sample to neutralize the stable free radical DPPH • (antiradical activity - ARA) is defined as: ARA = (A-B) / A × 100%, where A is the light absorption of the control, B is the light absorption of a sample (2 hours after mixing withe working solution of DPPH •). Chlorogenic acid is taken as the standard of antioxidant activity. TAOA is expressed in mg of chlorogenic acid per 1 g of a sample. If seed are noticeable for very high TAOA, the amount of an extract to be added to DPPH solution should be decreased, for example, 0.2 ml. In this case, a new calibration curve of the standard is plotted.

Conclusions. In recent years, the trend in use of methods for determining antioxidant activity has been gradually shifting towards the development and use of simple and inexpensive methods of evaluation of a large number of biological samples. This is attributed not only to the growing interest both of nutritionists and of dietitian to the search for new sources of valuable functional food products but also to the necessity of screening the plant gene pool in order to identify the most promising for biofortification accessions