Question

1. Propose a method to test the antioxidant activity (DPPH Scavenging method) of organic compounds. Write a detailed report about the protocol. Write the purpose, materials, calculations, methods, etc.

Answer 1

Solution:-

Purpose:-

α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method offers the first approach for evaluating the antioxidant potential of a compound, an extract or other biological sources. This is the simplest method, wherein the prospective compound or extract is mixed with DPPH solution and absorbance is recorded after a defined period.

Thus it is important to know the antioxidant content and their efficacy in foods, for preservation or protection against oxidative damage, to avoid deleterious changes and loss of commercial and nutritional value This necessitates the development of a rapid method for determining the potential antioxidant capacity in various foods.

DPPH Scavenging method:-

DPPH assay is considered a valid accurate, easy and economic method to evaluate radical scavenging activity of antioxidants, since the radical compound is stable and need not be generated. Sanchez-Moreno proposed a new methodology for the evaluation of antiradical efficiency towards DPPH, which is advantageous over other methods. It considers not only the antioxidant concentration but also the reaction time of scavenging reaction to reach the plateau. The results are highly reproducible and comparable to other free radical scavenging methods. The antioxidant efficiency is measured at ambient temperature so that the risk of thermal degradation of the molecules tested is eliminated . In optothermal window mode, the method can also be utilized for the evaluation of antioxidant efficiency of the compounds, which form opaque solution. It can also be used for online monitoring of changes in food containing natural antioxidants.

Calculation:-

The antioxidant activity is calculated by determining the decrease in the absorbance at different concentration by using the equation.

%Antioxidant activity = [E(_radical)-E_(standard)/ E(_radical)]

where E is the extinction coefficient of DPPH.

Material;-

Complex mixtures such as plant extracts, the results should be expressed as DPPH equivalents per gram of material. Suitable standards, like ascorbic acid and α-tocopherol could also be used to check the correctness of the procedures