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Determination of Antioxidant Activity through DPPH Assay


Antioxidants are compounds that protect cells from oxidative stress caused by free radicals. The DPPH assay is a widely used method to determine the antioxidant activity of plant extracts. |

A. Definition of DPPH Assay

The DPPH assay, or 2,2-diphenyl-1-picrylhydrazyl assay, is a widely used method to assess the antioxidant capacity of a substance. It measures the ability of a sample to neutralize free radicals by donating hydrogen atoms.

B. Importance of Antioxidant Activity Assessment

Antioxidants play a pivotal role in safeguarding cells from oxidative stress, contributing to overall health. The assessment of antioxidant activity is crucial in understanding the potential benefits of various substances.


In the DPPH assay, lies a stable free radical, DPPH, characterized by its deep purple color. When an antioxidant encounters DPPH, it donates a hydrogen atom, causing the DPPH radical to be reduced to its non-radical form, DPPH-H. This reduction is accompanied by a loss of color, transitioning from purple to a pale yellow hue. The extent of decoloration serves as a direct measure of the antioxidant’s ability to scavenge free radicals.

Significance of Antioxidant Activity

Antioxidants play a pivotal role in maintaining overall health by actively combating oxidative damage at the cellular level. This is particularly significant in the context of DPPH assays, as they provide a means to assess the effectiveness of substances in countering oxidative stress.

A. Role of Antioxidants in Health

The human body is constantly exposed to environmental factors such as pollution, UV radiation, and even the byproducts of normal metabolic processes. These factors generate free radicals—highly reactive molecules that can cause oxidative damage to cells. This damage is implicated in various health conditions, including inflammation, cardiovascular diseases, and even the aging process.

Antioxidants, acting as a defense mechanism, neutralize these free radicals by donating electrons without becoming destabilized themselves. This protective role is crucial for maintaining the integrity and functionality of cells, tissues, and organs. In essence, antioxidants act as cellular bodyguards, preventing the destructive chain reactions initiated by free radicals.

The significance of antioxidant activity in health is further emphasized by its association with the prevention of chronic diseases. Studies have linked a diet rich in antioxidants to a reduced risk of conditions such as cancer, neurodegenerative disorders, and cardiovascular diseases. Therefore, understanding and measuring antioxidant activity, as facilitated by DPPH assays, becomes integral in assessing the potential health benefits of various substances.

B. Connection to Free Radicals

To grasp the importance of antioxidant activity, it is essential to comprehend the role of free radicals in cellular damage. Free radicals are highly reactive molecules with unpaired electrons, making them unstable and prone to “stealing” electrons from other molecules in an attempt to stabilize themselves. This process, known as oxidative stress, sets off a chain reaction that damages cellular structures, including proteins, lipids, and DNA.

Oxidative stress is implicated in the aging process and the development of various diseases. Free radicals can lead to inflammation, tissue damage, and mutations, contributing to conditions such as cancer and neurodegenerative disorders. The body’s natural defense against oxidative stress is the presence of antioxidants, which neutralize free radicals and prevent their harmful effects.

The DPPH assay is a valuable tool in quantifying a substance’s ability to counteract free radicals. By measuring the substance’s capacity to reduce the DPPH radical, the assay provides a quantitative assessment of its antioxidant potential. This not only aids researchers in understanding the efficacy of specific compounds but also allows for comparisons between different substances in terms of their ability to neutralize free radicals.

Applications of DPPH Assay

A. Pharmaceutical Industry

In drug development, assessing the antioxidant activity of compounds is crucial. DPPH assays assist pharmaceutical researchers in identifying potential candidates for further exploration.

B. Food and Beverage Sector

The food industry benefits from DPPH assays to evaluate the antioxidant content of various products. Consumers, too, gain insights into the health benefits of their food choices.

C. Cosmetics and Skincare Products

Antioxidant-rich skincare products are in high demand. DPPH assays help manufacturers validate the efficacy of these products, ensuring they deliver the promised benefits.

Step by Step procedure-

  1. Sample Preparation
    Prepare the sample using the same procedure as discussed in the post Determination of Total Phenolic Content (TPC) in Food: All in 1 Comprehensive Guide in Procedure section.
  2. Collect 100μL of the supernatant from the above step in test tubes.
  3. Add 3.9mL DPPH solution having the concentration of 6×10-5 mol/L
    How to prepare DPPH solution having the concentration of 6×10-5 mol/L ?
    (Add 4mg of DPPH powder in 100mL of Distilled Water)
  4. Incubate the solution for 30min in dark at ambient temperature.
  5. Record the absorbance at 515nm
  6. First Put Pure Methanol in both the cuvettes for blank setting
    Then Measure Absorbance of DPPH solution of Control solution prepared in step-3, and name it as Acontrol
  7. Now measure the Absorbance value of the samples and name it as Asample

Calculating Antioxidant Activity

The antioxidant activity is calculated using the following formula:

Antioxidant Activity (%) = [(Acontrol - Asample) / Acontrol] x 100


  • Acontrol is the absorbance of the blank DPPH solution
  • Asample is the absorbance of the incubated reaction mixture

A higher percentage value indicates stronger antioxidant activity.

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