Dear M. Mallique Qader,

Please read the following text:

Nitric Oxide Radical Scavenging Assay

The assay is the nitric oxide radical scavenging assay [24]. The extracts were prepared from a 10 mg/mL ethanol crude extract. These were then serially diluted with distilled water to make concentrations from 100−1000 μg/mL of the three plants and the standard gallic acid. These were stored at 4°C for later use. Griess reagent was prepared by mixing equal amounts of 1% sulphanilamide in 2.5% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in 2.5% phosphoric acid immediately before use. A volume of 0.5 mL of 10 mM sodium nitroprusside in phosphate buffered saline was mixed with 1 mL of the different concentrations of the ethanol extracts (100−1000 μg/mL) and incubated at 25°C for 180 mins. The extract was mixed with an equal volume of freshly prepared Griess reagent. Control samples without the extracts but with an equal volume of buffer were prepared in a similar manner as was done for the test samples. The colour tubes contained ethanol extracts at the same concentrations with no sodium nitroprusside. A volume of 150 μL of the reaction mixture was transferred to a 96-well plate. The absorbance was measured at 546 nm using a SpectraMax Plus UV-Vis microplate reader (Molecular Devices, GA, USA). Gallic acid was used as the positive control. The percentage inhibition of the extract and standard was calculated and recorded. The percentage nitrite radical scavenging activity of the ethanol extracts and gallic acid were calculated using the following formula:

Nitic Oxide Scanenge (%) = A control - A test/A control x 100

percentage nitrite radical scavenging activity:where = absorbance of control sample and = absorbance in the presence of the samples of extracts or standards.

http://www.hindawi.com/journals/jfp/2014/918018/

Hoping this will be helpful,

Rafik

Dear Professor Rafik Karaman ,

I can intervene in this discussion.

I have the same problem with this mannipulation.

I practiced this method but the problem I noticed $ the decrease in percent inhibition as a function of increasing concentrations.

I have not found the resolution of my problem but I think it is the blank that is formed by the distilled water.

Please answer me

Sorry to say but the attached research article has no such assay mentioned. Can you please give any reference for the said protocol.

Dear Zeenat Ayoub;

There is below the protocol of this method:

Please help me to solve the problem of my experiment concerning the anti-inflammatory activity with the Griess reagent and sodium nitroprusside.

I repeated the experiment more than a month but I find that the percentages of inhibition decrease with the increase of the concentrations.

I prepared the Griess reagent as follows;

1% sulphanilamide in 2.5% orthophosphoric acid, .1% N- (1-naphthyl) ethylenediamine dihydrochloride in distilled water.

both solutions are kept separately. I mixed volume / volume during the use

The protocol:

0.5ml extracts (different concentrations from a stock solution of 1mg / ml) + 0.5 ml sodium nitroprusside (10mM in phosphate buffered saline (pH 7.4)

incubation at 25 ° C for 2.5h.

then I added 1ml of Griees reagent (1ml), incubate 30min at 25 ° C.

Read the absorbances at 546nm of the tests and the control against the blanc which is formed of the distilled water.

Here are the values ​​obtained:

C (mg / ml) % inhibition

1 4.373698304

0.5 10.7606863

0.25 16.05672915

0.125 15.54100962

With all my thanks.

Same is the case with me