Bangladesh J Pharmacol. 2016; 11: 570-572 

VISUAL EXPERIMENT 

Ferric reducing anti-oxidant power assay in plant extract 

Maruthamuthu Vijayalakshmi and Kandasamy Ruckmani 

Department of Pharmaceutical Technology, Bharathidasan Institute of Technology campus, Anna University, 

Tiruchirappalli 620-024. India. 

Correspondence to Kandasamy Ruckmani at hodpharma@gmail.com 

First Published: 1 June, 2016 

DOI: 10.3329/bjp.v11i3.27663 

ABSTRACT

The ferric reducing anti-oxidant power (FRAP) assay involved the following steps:  a) preparation of 
samples, b) reactions and c) finally measuring absorbance of sample and standard at 700 nm using 
spectrophotometer. The samples were methanolic extract, different fractions (n-hexane, chloroform) and 
standard ascorbic acid. The solutions prepared were buffer solution (pH 6.6), 1% potassium ferricyanide 
solution, 10% trichloroacetic acid and 0.1% ferric chloride.   

INTRODUCTION

The total antioxidant activity can be measured by the ferric reducing antioxidant power assay (FRAP). The 
flavonoids and phenolic acids are present in the medicinal plant exhibit strong antioxidant activity which 
is depending on their potential to form the complex with metal atoms, particularly iron and copper. This 
method is based on the principle of increase in the absorbance of the reaction mixtures, the absorbance 
increases the antioxidant activity increases. The antioxidant compound present in the samples forms a 
colored complex with potassium ferricyanide, trichloroacetic acid and ferric chloride, which is measured 
at 700 nm by UV-Spectrophotometer.  

MATERIALS AND EQUIPMENTS

 Deionized water

 Potassium ferricyanide

 Sodium chloride

 Potassium chloride

 Disodium hydrogen phosphate

 Potassium dihydrogen phosphate

 Hydrochloric acid

 Trichloroacetic acid

 Ferric chloride

 Centrifuge tubes

 Pipette

 Water bath

 Vortex shaker

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 Centrifuge

 UV-Spectrophotometer

PREPARATION OF REAGENTS 

 0.2M phosphate buffer (pH 6.6):  8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium
hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate was taken in a 1,000 mL standard flask
and add 800 mL of distilled water and adjust the pH 6.6 using hydrochloric acid and adjust the volume
with deionised water.

 Potassium ferricyanide (1%): 1 g of potassium ferricyanide was dissolved in 100 mL of deionised water.

 Trichloroacetic acid (10%): 10 g of trichloroacetic acid was dissolved in 100 mL of deionised water.

 Ferric chloride (0.1%): 100 mg of ferric chloride was dissolved in 100 mL of deionised water.

 Ascorbic acid (0.1%): 1 mg of ascorbic acid was dissolved in 1 mL of water.

VIDEO CLIP 

Ferric reducing anti-oxidant power assay: 21 min 18 sec 

METHOD 

 Different concentrations of the methanolic extract of M. serratulum and its various fractions (10-50
μg/mL) was added to 2.5 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium
ferricyanide [K3Fe(CN)6] solution.

 The reaction mixture was vortexed well and then incubated at 50°C for 20 min using vortex shaker.

 At the end of the incubation, 2.5 mL of 10% trichloroacetic acid was added to the mixture and
centrifuged at 3,000 rpm for 10 min.

 The supernatant (2.5 mL) was mixed with 2.5 mL of deionised water and 0.5 mL of 0.1% ferric chloride.

 The colored solution was read at 700 nm against the blank with reference to standard using UV-
Spectrophotometer. Here, ascorbic acid was used as a reference standard, the reducing power of the
samples were comparable with the reference standard.

Bangladesh J Pharmacol 2016; 11: 570-572 571 

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DISCUSSION 

In this study, the reducing capacity of the extracts and fractions were performed using Fe3+ to Fe2+ 

reduction assay.  In this experiment, the yellow color changes to pale green and blue color depending on 

the concentration of antioxidants in the samples. The antioxidants such as phenolic acid sand flavonoids 

were present in considerable amount in the extract of M. serratulum. All the samples showed reducing 

capacity in a concentration dependant manner. 

 

REFERENCES 

Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Jpn J Nutr. 1986; 44: 
307-15. 

Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, Ju YH. Effect of 
extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila 
aromatica. J Food Drug Anal. 2014; 22: 296-302.   

 

  

PRECAUTION 

The solution must be prepared on the day of experiment and should be protected from sunlight. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

572 Bangladesh J Pharmacol 2016; 11: 570-572  



 

 

 

 

 

 

 

 

 

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