REDUCING POWER ASSAY

PRINCIPLE

Reducing power is associated with antioxidant activity and may serve as a

significant reflection of the antioxidant activity. Compounds with reducing power indicate
that they are electron donors and can reduce the oxidized intermediates of lipid
peroxidation processes, so that they can act as primary and secondary antioxidants. In
this assay, the yellow colour of the test solution changes to various shades of green and blue depe
nding on the reducing power of each compound. Presence of reducers causes the conversion of th
e Fe3+/ferricyanide complex used in this methodto the ferrous form. By measuring the formation
of Pearl’s Prussian blue at 700nm, it is possible to determine the concentration of Fe3+ ion. Red
ucing power assay method is based on the principle that substances, which have reduction
potential, react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+),
which then reacts with ferric chloride to form ferric– ferrous complex that has an absorption
maximum at 700 nm.

Potassium ferricyanide + Ferric chloride Potassium ferrocyanide+Ferrouschloride

PROCEDURE

According to this method, 1 ml methanolic extract (40 to 200 μg/ml) were mixed with 2.5
ml of (pH 6.6) phosphate buffer and 2.5 ml of (1%) potassium ferricyanide. The mixture was
incubated at 50°C in water bath for 20 min. After cooling, aliquots of 2.5 ml of (10%)
trichloroacetic acid were added to the mixture, which was then centrifuged at 3000 rpm for 10
min. The upper layer of solution 2.5 ml was mixed with 2.5 ml distilled water and a freshly
prepared 0.5 ml of (0.1%) ferric chloride solution. The absorbance was measured at 700 nm in
UV spectrometer. Ascorbic acid at various concentrations (10 to 100 μg/ml) was used as
standard.

Absorbance of control−Absorbance of sample

%RSA= × 100
Absorbance of control
Reference = Ascorbic acid
Wavelength = 700nm
Blank = Phosphate buffer pH 6.6
Control = 1 ml methano l+ 2.5 ml phosphate buffer pH 6.6 + 2.5 ml 1% Potassium
ferricyanide + 2.5 ml Trichloroacetic acid followed by centrifugation at 3000 rpm for 10 min. 2.5
ml of Supernatent+2.5 ml distilled water + 0.5 ml 0.1% ferric chloride solution.

REAGENTS

1) 1% Potassium ferricyanide
Take 1 g of Potassium ferricyanide and dissolve it in distilled water, made up to 100 ml.
2) 10% Trichloroacetic acid
Take 10 g of Trichloroacetic acid and dissolve in distilled water, made up to 100 ml.
3) 0.1% Ferric chloride
Take 0.1g of Ferric chloride and dissolved in 100ml distilled water.
4) Phosphate buffer pH 6.6
Take 800ml of distilled water in a container. Add 10.761g of Sodium phosphate dibasic
heptahydrate to the solution. Add 8.26g of Sodium phosphate monobasic monohydrate to the
solution. Adjust solution to final desired pH using Hydrochloric acid or Sodium hydroxide.

STANDARDIZATION OF REDUCING POWER ASSAY

RESULT ANALYSIS
Percentage inhibition activity of Ascorbic acid
Trial done date : 10-3-202
Blank : Phosphate buffer pH 6.6
Control : 1 ml methanol+2.5 ml phosphate buffer pH 6.6+2.5 ml 1%
Potassium ferricyanide+2.5 ml Trichloroacetic acid followed by centrifugation at 3000
rpm for 10 min. 2.5 ml of Supernatent+2.5 ml distilled water+ 0.5 ml 0.1% ferric
chloride solution.
 Reducing power assay of Ascorbic
acid
0.7Concentration % Inhibition
y
10 0.275
= 0.0021x +
0.2463
50 0.331
Absorbance
0
.
3 100 Linear
0.464
0 150 0.577
.
1
0 200 5 1 1 2 2 0.655
Concentration

Discussion
Reference literature depicts that reducing power is a function of concentration.