Quantitative Test of the Antioxidant Activity (Frap Method)

3.2 Quantitative Test of the Antioxidant Activity (Frap Method)

3.2.2 Reagent preparation:

1. 300 mmol/L acetate buffer, pH 3.6 (3.1 g Sodium acetate 3 H 2 O + 16 ml acetic acid per Liter of buffer solution)

2. 10 mmol /L TPTZ in 40 mmol/L HCL = 10 x 312.34 mmol = 3123.4 ppm.

3. 20 mmol/L FeCl 3 .6H 2 O = 20 x 270.5 mg/mmol = 5410 mg/L

FRAP reagent was prepared by mixing 25 ml acetate buffer + 2.5 ml TPTZ solution and 2.5 ml Ferric chloride solution. TPTZ trihydrate in aqueous ethanol, reacts with ferrous ion to yield intense violet color over pH range 3.4- 5.8 with maximum

absorption (Fe(TPTZ) 2+

2 (water) at 593 nm ( 22,600). At low pH the reduction of iron (III) tripyridyltriazine (FeIII TPTZ) is carried out to become iron (II) (Fe IITPTZ), which can be observed from color changes to blue intensive color. This color is measured at of 593 nm. The change of absorbances is in accordance with the antioxidant activities.

The sample was dissolved in methanol in a concentration of 50 g/mL then FRAP solution (50 g/mL was added (volume of sample: volume of FRAP solution = 1:1).

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

The mixture was incubated for 20 min and the absorbances were measured at 593 nm.

The antioxidant activity was measured as the percent capacity of the sample and is calculated using the equation:

Eq.2

Note : T -As

S = Transmittance of FRAP solution after the adding of the test sample: T S = 10

A S = Absorbance of FRAP solution after the adding of the sample .

3.2.3 Quantitative Analyses of the Sample by FRAP’s Method

50.0 mg of each extract was weighed and dissolved in about 5 ml MeOH in a sonicator. Then it was filtered to 10.0 ml volume flask and MeOH was added to make

10.0 ml ( = 5000 ppm) From this solution 5 more dilutions were made: 2.5 ml solution was pipetted into a volume flask and MeOH was added to make 5.0 ml solution (2500 ppm). 1.0 ml solution was pipetted into a volume flask and MeOH was added to make 5.0 ml solution (1000 ppm). 1.0 ml solution was pipetted into a volume flask and MeOH was added to make 10.0 ml solution (500 ppm). 0.25 ml solution was pipetted into a volume flask and MeOH was added to make

5.0 ml solution (250 ppm). 0.25 ml solution was pipetted into a volume flask and MeOH was added to make 10.0 ml solution (125 ppm).

Then 2.0 ml of Frap solution was added to 2.0 ml sample solution, the absorbance was measured, and then the T was calculated.

The absorbance was measured at 593 nm with MeOH:distilled water: HCl 0.04 M as the blank. Incubation time was 20 minutes in the dark.

Table 3. Antioxidant capacity of red fruit products by FRAP Method

No DIC-assisted

Conv. Drying Oil Ethanol Ext

Conv. Drying

DIC-assisted

Hexane extr 1 Y=

Ethanol Ext

Hexane extr

0.104 x+12.32 0.082x+7.103

0.080x+5.670

0.034x+8.186 Cannot be

A 50 (ppm) 362.31

1229.82 analyzed by FRAP

2 Y= 0.105 x + 12.01 0.076 x + 8.452 0.085 x + 4.503 0.034 x + 5.680 method. A 50 (ppm) 361.81

1303.53 Av of A 50 ppm) 362.06±0.35

The oil could not be analyzed by FRAP method because the addition of FRAP reagent caused the formation of precipitate, which disable the reading in the Spectrophotometer.

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a