Antioxidant Assays: 1. DPPH Assay (2, 2-Diphenyl-1-Picrylhydrazyl)
Antioxidant Assays: 1. DPPH Assay (2, 2-Diphenyl-1-Picrylhydrazyl)
Antioxidant Assays: 1. DPPH Assay (2, 2-Diphenyl-1-Picrylhydrazyl)
Where, RSA is the Radical Scavenging Activity; Abs control is the absorbance of DPPH
radical + ethanol; Abs sample is the absorbance of DPPH radical + plant extract.
2 Phosphomolybdenum assay
The antioxidant activity of samples was evaluated by the green phosphomolybdenum
complex formation according to the method of Prieto (1999).
Principle
This
method
is
based
on
the
reduction
of
phosphomolybdic
acid
to
Reagent preparation
156l NBT was prepared in 10ml of 100mM phosphate buffer (pH 8).
468M NADH solution was prepared in 10ml of 100mM phosphate buffer (pH 8).
60M PMS in 10ml of 10mM PO4 buffer pH-8.
10mg extract in 0.1ml DMSO+ 0.9 ml PO4 buffer.
Working procedure
1ml of NBT solution, 1ml of NADH solution, 0.1ml of plant extract (10mg in 0.1ml
DMSO and 0.9ml PO4 buffer) and 0.1ml of PMS solution were added together and incubated
at 25C for 5 min. After 5 min the absorbance was read at 560 nm.
4 Hydroxyl radical scavenging activity
Hydroxyl radical is one of the potent reactive oxygen species in the biological system.
It reacts with polyunsaturated fatty acid moieties of cell membrane phospholipids and causes
damage to cell (Halliwell and Gutteridge, 1981).
Principle
HRS assay is used to find the scavenging activity of free hydroxyl radicals (which
damage the body cells) like hydrogen peroxide in the presence of different concentrations of
plant sample. The model used is ascorbic acid-iron-EDTA model of hydroxyl radical
generating system. This is a totally aqueous system in which ascorbic acid, iron and EDTA
conspire with each other to generate hydroxyl radicals.
Reagent preparation
Iron-EDTA solution was prepared by mixing 0.13% ferrous ammonium sulphate and
0.26% EDTA.
0.018% EDTA was prepared by dissolving 0.018g EDTA in 100ml dist. H2O.
0.22% ascorbic acid was prepared by dissolving 0.22g ascorbic acid in 100ml dist. H2O.
17.5% TCA was prepared by dissolving 17.5g TCA in 100ml of dist.H2O.
Nash reagent was prepared by adding 7.5g ammonium acetate, 0.5ml of glacial acetic acid
and 0.2ml of acetone to 100ml dist.H2O.
Working procedure
Various concentrations of extract (250, 500, 750, 1000g) were taken and 1ml of iron
EDTA solution, 0.5ml of EDTA solution, 1ml of DMSO and 0.5ml of ascorbic acid were
added to it. The mixture was incubated in a boiling water bath at 80 to 90C for 15 min. After
incubation, 1ml of ice cold TCA and 3ml of Nash reagent were added and the reaction
mixture was incubated at room temperature for 15 min. The absorbance was read at 412 nm.
The % hydroxyl radical scavenging activity is calculated by the following formula,
Where, HRSA is the Hydroxyl Radical Scavenging Activity, Abs control is the absorbance of
control and Abs sample is the absorbance of the extract.
5 Metal chelating activity
Principle
Ferrozine can quantitatively chelate with Fe2+ and form a red coloured complex. This
reaction is limited in the presence of other chelating agents and results in a decrease of the
red colour of the ferrozine-Fe2+ complex. Measurement of the color reduction estimates the
chelating activity to compete with ferrozine for the ferrous ions (Soler-Rivas et al., 2000).
The antioxidants present in plant extract forms a coordinate complex with the metal ions
(chelating activity) and inhibit the transfer of electrons. Thus oxidation reaction is arrested
and no free radicals are produced.
Reagent preparation
2mM ferrous chloride was prepared by dissolving 2.5mg of FeCl2 in 10ml of dist.H2O.
5mM ferrozine was prepared by dissolving 24mg in 10ml of dist.H2O.
Working procedure
100l of plant extract (10mg in 1ml DMSO) was added to 50l of 2mM ferrous
chloride and 200l of 5mM ferrozine solution. The solution was mixed thoroughly and
incubated in dark at room temperature for 10 min. The absorbance was read at 562 nm. 100l
EDTA (10mg/1ml DMSO) was used as standard.