Radical Scavenging Activity

Assessment of the Radical Scavenging Activity of Plant Extracts and Fractions Using DPPH Chemical Assay

Background

2,2-Diphenyl-1-picrylhydrazyl radical (DPPH^.) is a stable free radical which can be used to assess the radical scavenging activity of plant materials. At radical state, the methanolic solution of this compound is purple (absorbs light at wavelength of 540 nm) which when reacts with an antioxidant (e.g, flavonoid) it is reduced to the molecular form (DPPHH) which is yellow with no absorbance at 540 nm, Fig. 1. This change in absorbance can be a measure to the radical scavenging power of the test sample.

Fig (1) Principle of DPPH Assay, adapted from Amic et al.(2003).

Trolox^® (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, Fig. 2) is a vitamin E derivative and is known with its potent radical scavenging activity, it as been used as positive control or assay calibrator.

Fig. 2 Trolox^® (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) adapted from Sigma-aldrich.com

Reagents preparation

Test samples: Dissolve the plant extract at the agreed concentration range as 10X stocks in methanol.

DPPH radical solution: Dissolve 4mg DPPH in 100 ml Methanol (0.004% solution), store in dark until use.

Trolox solution: Prepare different concentrations of 10X trolox in methanol (50-800µM).

Design 96-well plate layout.

Method

All samples, blank and calibrators are to be added in triplicates.

In each well of a 96-well plate, add each sample concentration from the stock as 20µl

Use the solvent (20 µl) as a blank.

For calibration curve use 20 µl of 10X TROLOX^® stock solutions

Start the reaction by addition of 180µl DPPH radical solution, start time for 15 minutes from the first addition, incubate at room temp.

Measure the absorbance of each well at 540 nm (Nara, Miyoshi et al. 2006).

The radical scavenging activity can be measured for sample and calibrator from the

following equation:

% inhibition= 100X (A_blank-A _sample)

A _blank

where A_blank= absorbance of blank, A_sample = absorbance of sample

From the TROLOX^®calibration curve, measure the Trolox equivalent antioxidant capacity (TEAC) of samples.

References:

Amic, D et al. (2003). "Structure-Radical Scavenging Activity Relationships of Flavonoids. "Croatica Chemica Acta 76 (1) 55-61.

Nara, K., T. Miyoshi, et al. (2006). "Antioxidative activity of bound-form phenolics in potato peel." Biosci Biotechnol Biochem 70(6): 1489-91.

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