Testing Antioxidant Activity by Using DPPH (1,1-diphenyl-2-pikrilhidrasil) Method

Various methods of measuring antioxidant activity has been used to monitor and compare the antioxidant activity in food. Several years ago, testing and testing of oxygen radical absorbance chemiluminance used to evaluate food antioxidant activity, serum and fruits. This type of method used to determine the antioxidant activity of foods that involve methods of electron spin resonance (ESR). This analysis method to measure the antioxidant activity by capturing free radicals such as radical 1.1-diphenyl-2-pikrilhidrasil (DPPH), superoxide anion radicals (O2), hydroxyl radical (OH) or peroxy radicals (ROO^●) (Prakash, 2001).
             Tests for antioxidant activity by free radicals is a method that is fast, simple and easy. Radical ABTS [2,2-azinobis (acid 3 - etilbenzotiazolin-6-sulfonate)] was used to test the free radical capturing ability of flavonoids and phenolic through nature as giver of H atoms or electrons. Another method is a method using free radical DPPH (1,1-diphenyl-2-pikrilhidrasil). DPPH method is accurate and fast method (Prakash, 2001).

 The antioxidant activity can be measured by various parameters, including inhibition of oxidation rate and the reference standard. One standard reference is the acid 6-hydroxy-2 ,5,7,8-tetrametilkroman-2-carboxyl (Trolox), ascorbic acid (vitamin C) and α-tocopherol (vitamin E) (Prakash, 2001 and Molyneux, 2003) .

 DPPH is widely used to test the ability of compounds that act as electron donors or hydrogen, and to evaluate the antioxidant activity in food systems. This method also has been used several years ago to know the amount of antioxidants in complex biological systems. DPPH method is not specific to a certain antioxidant components, but for all the antioxidant compounds in the sample. Measurement of total antioxidant capacity will help to understand the functional properties of foods (Prakash, 2001).

 Method of DPPH (1,1-Diphenyl-2-picrylhydrazyl), which has been developed by Hatano, et al (1998) and Amarowicz, et al (2000), is used to determine the ability of antioxidants to capture free radicals. Calculation based on the value of absorbance at 517 nm. DPPH is a stable free radical. Since DPPH accept an electron or hydrogen radical to become a stable magnetic molecules. Reduce the ability of DPPH radical is known through a reduction in absorbance at 517 nm.

Antioxidant activity of samples can be expressed as mikromolekul Trolox equivalent (TE) per 100 gram sample or TE/100 g (Prakash, 2001). The antioxidant activity also can be expressed with units of% activity. Absorbance of control used in this procedure is the absorbance of DPPH DPPH, while the blank used is ethanol. Based on these formulas, the greater the level of discoloration (absorbance of the smaller) the higher the value the arrest of radical activity (Molyneux, 2003).

In the DPPH method are the parameters nDPPH and efficient concentration or EC₅₀ values. NDPPH parameter is the number of DPPH molecules reduced by one molecule of substrate. This shows antivitas antioxidants associated with the molecular structure of the substrate. While the EC₅₀ value is defined as the concentration of substrate that causes loss of activity of DPPD (color) as much as 50%. EC₅₀ parameter to show antioxidant activity, the lower the EC₅₀ value, the higher the antioxidant activity (Molyneux, 2003).
           The antioxidant activity of a food can vary when tested with different methods. Unlike the DPPH method that measures the total antioxidant activity, several other methods are limited to measuring the component soluble in solvents used in the analysis. DPPH method measures all components of antioxidants, either fat soluble or water.