4.4. DPPH Assay

DPPH Radical Scavenging Assay for Evaluation of Antioxidant Activity in Plant Extracts

Abstract

This protocol describes the determination of antioxidant activity using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The method is based on the reduction of DPPH radicals by antioxidants, resulting in a color change from purple to yellow, which is measured spectrophotometrically at 517 nm. The antioxidant capacity is expressed as percentage inhibition and IC₅₀ values.

Materials and Reagents

• DPPH (2,2-diphenyl-1-picrylhydrazyl) 
• Methanol (analytical grade) 
• Dimethyl sulfoxide (DMSO) 
• Gallic acid (standard antioxidant) 
• Plant extracts (e.g., Litsea martabanica root extract) 

Equipment

• 96-well microplate 
• Micropipettes and tips 
• Volumetric flask (100 mL) 
• Microplate reader (set at 517 nm) 
• Vortex mixer (optional) 

Reagent Preparation

1. DPPH Solution

• Dissolve 0.0066 g DPPH in methanol and adjust the volume to 100 mL. 
• Measure absorbance at 517 nm and adjust to 1.7–2.0 before use. 

2. Gallic Acid Stock Solution

• Dissolve 10 mg gallic acid in 1 mL DMSO to obtain a stock solution (10 mg/mL). 

Procedure

A. Preparation of Samples and Standards

• Dilute plant extracts to the required concentrations: 
  • Nutmeg extract: 25–800 µg/mL 
  • Haem extract: 321.5–10,000 µg/mL 
• Dilute gallic acid to 0.9375–30 µg/mL. 

B. DPPH Assay

• Divide a 96-well plate into five sections: 
  • Sample test 
  • Sample blank 
  • Standard test 
  • Reagent blank (0% inhibition) 
  • Blank 
• Add 20 µL of sample solutions into designated wells (test and sample blank). 
• Add 20 µL of gallic acid into standard wells. 
• Add 20 µL methanol into reagent blank wells. 
• Add 180 µL DPPH solution into: 
  • Sample test wells 
  • Standard wells 
  • Reagent blank wells 
• Add 180 µL methanol into sample blank wells. 
• Add 200 µL methanol into blank wells. 
• Mix gently and incubate at room temperature in the dark for 30 min. 
• Measure absorbance at 517 nm using a microplate reader. 
• Perform all experiments in triplicate. 

Data Analysis

The percentage of DPPH radical scavenging activity (% inhibition) is calculated using the following equation:

• Results are expressed as mean ± SD from three independent experiments. 
• Plot % inhibition versus concentration. 
• Determine IC₅₀ values using linear regression analysis. 

Notes

• The standard wavelength for DPPH assay is 517 nm, corresponding to the maximum absorbance of DPPH radicals. 
• If the instrument does not support 517 nm, an alternative wavelength (e.g., 492 nm) may be used, provided that measurements are performed consistently and appropriate calibration is applied. 
• Prepare DPPH solution freshly or store in the dark to maintain stability. 
• Maintain consistent incubation time across all samples. 
• Avoid light exposure during incubation to prevent DPPH degradation. 
• Use the same solvent system for samples and controls to minimize interference. 

Acknowledgments

This protocol was adapted from our published work on the antioxidant activity and hepatoprotective effects of Litsea martabanica root extract.

Competing Interests

The authors declare no competing interests.

Keywords

DPPH assay; antioxidant activity; free radical scavenging; plant extracts; gallic acid; IC₅₀; spectrophotometry