Nitric Oxide Scavenging Activity

Nitric Oxide radical inhibition estimated by the use of Griess Illosvoy reaction (Garrat, 1964). Nitric oxide generated from sodium nitroprusside and measured by the Griess reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitric ions that can be estimated by use of Griess reagent. Scavenger of nitric oxide competes with oxygen leading to reduced production of nitric oxide. Sodium nitroprusside (5 mM) in phosphate–buffered saline (PBS) was mixed with 3.0ml of different concentrations (10-100μg/ml) of the drugs dissolved in the suitable solvent systems and incubated at 25⁰C for 150 min.

The samples from the above were reacted with Griess reagent (1% sulphanilamide, 2%H3PO4 and 0.1% napthylethylenediamine dihydrochloride).

The absorbency of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546nm

Griess Reagent:

Physical Appearance: Colorless to lightly pink liquid

Description: Reagent for the one step determination of nitrite (NO2-) in biological media.

Form: 1 part of 0.1% naphthyl ethylenediamine dihydrochloride in distilled water plus 1 part 1% sulfanilamide (or sulfanilic acid) in 5% concentrate H3PO4.

Prolong Storage: Keep cool and dry at +4^oC

Principle: This assay relies on a diazotization reaction that was originally described by Griess in 1879. Modifications have been made to the original reaction through the years. This procedure is based on the chemical reaction shown below, which uses sulfanilamide and naphthylethylenediamine dihydrochloride (NED) under acidic conditions. Sulfanilamide and NED compete for nitrite in the Griess reaction. This reagent detects NO₂^- in a variety of biological and experimental liquid samples such as plasma, serum, urine and tissue culture medium. The limit of detection is 2.5 uM (125 pmol) nitrite (in distilled, deionized water); however, the sensitivity will vary depending upon the sample used. Individual researchers must determine the limits for their individual experiments and referred to the absorbance at standard solutions of potassium nitrite, treated in the same way with Griess reagent.

The percentage scavenging of nitric oxide of Plant extract and standard compounds was calculated using the following formula:

NO Scavenged (%) = (A cont - A test)/A cont × 100

Where A cont is the absorbance of the control reaction and

 A test is the absorbance in the presence of the sample