Tests

How to determine Antioxidant potential of plant extract?

Antioxidants are those types of molecules that neutralize the harmful compounds called free radicals that damage living cells, spoil food, and degrade materials such as rubber, gasoline, and lubricating oils. Antioxidants arrest the chain reaction initiated by free radicals. Some antioxidants donate electrons to stabilize and neutralize the dangerous free radicals. Other antioxidants work against the molecules that form free radicals, destroying them before they can begin the domino effect that leads to oxidative damage.

There are several ways for the assessment of antioxidant potential of plant extract_

DPPH-free radical scavenging assay:

The DPPH antioxidant assay is based on the ability of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron which is responsible for the absorbance at 517 nm, and also for visible purple yellow color. When DPPH accepts an electron donated ion by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the change in the absorbance.

To determine antioxidant potential of a plant extract, a suitably diluted sample solution is spotted on pre-coated silica gel of TLC plates. In order to resolve polar and non-polar components of the extract, the plates are developed in different solvent systems of different polarities (polar, medium polar and non-polar). Then the plates will be dried at room temperature and after drying they are sprayed with 0.02% 1-1diphenyl-2-picryl hydrazyl (DPPH) in ethanol. Bleaching of DPPH is visualized for a period of 10 minutes and then the color changes (yellow on purple background) will be remarked. When DPPH dissolves in ethanol, it will yield deep pink color. DPPH forms pale yellow or yellow color when it will be sprayed on the chromatogram of the extract which indicates the presence of antioxidants. 

Reducing power assay:

Different concentrations of the plant extract are mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of potassioum ferricyanide solution (1 %). After incubation period of 20 min at 50 °C, the mixtures are mixed with 2.5 mL of trichloroacetic acid (10 %) followed by centrifugation for 10 min, at 3000 rpm. Then 2.5 mL of the supernatant will be mixed with 2.5 mL of distilled H₂O and 0.5 mL of ferric chloride (0.1 %). Then the absorbance of this solution will be measured at 700 nm. Ascorbic acid served as positive control.