A new colorimetric DPPH• scavenging activity method with no need for a spectrophotometer applied on synthetic and natural antioxidants and medicinal herbs

Abstract

2,2-Diphenyl-1-picrylhydrazyl (DPPH^•) radical scavenging, the most commonly used antioxidant method with more than seventeen thousand articles cited, is very practical; however, as with most assays, it has the major disadvantage of dependence on a spectrophotometer. To overcome this drawback, the colorimetric determination of the antioxidant activity using a scanner and freely available Image J software was developed. In this new method, the mixtures of solutions of DPPH^• and standard antioxidants or extracts of common medicinal herbs were dropped onto TLC plates, after an incubation period. The spot images were evaluated with Image J software to determine CSC₅₀ values, the sample concentrations providing 50% colour reduction, which were very similar with the SC₅₀ values obtained with spectrophotometric method. The advantages of the new method are the use of lower amounts of reagents and solvents, no need for costly spectrophotometers, and thus significantly lowered costs, and convenient implementation in any environment and situation.

Keywords: 2,2-diphenyl-1-picrylhydrazyl; Image J software; TLC plates; antioxidant.

Figure 1.

(a) Concentration–absorbance graph used to calculate SC_50, (b) concentration–colour value graph used to calculate CSC₅₀ and (c) 1st degree derivative graph used to calculate CSC₅₀ for protocatechuic acid and ferulic acid standards.

Figure 2.

Linearity of SC₅₀ and CSC₅₀ values as a function of DPPH• radical concentration using Trolox standard.

Figure 3.

The effect of drop volume on colour value readings using Trolox standard.

Figure 4.

The effect of incubating the reaction mixtures in (a) test tubes and (b) on TLC for 55 min using shringaldehyde as standard, and (c) trends in absorbance decrease as DPPH was scavenged with the standards as a function of time, i.e. reaction kinetics.

Figure 5.

(a) Image of spots prepared with 1:1 serial dilution of gallic acid beginning from 3000 μg/mL highest concentration, and image of spots prepared with 1:1 serial dilution of alkanet extract beginning from 5000 μg/mL highest concentration containing (b) extract and reagent solvent (methanol) and (c) extract and DPPH^• reagent.

Figure 6.

The SC₅₀ and CSC₅₀ values of standards and extracts (showing a good correlation with R²=0.9923).