I have calculated nitric oxide scavenging activity with the help of griess reagent as follow:-
reagents
Phosphate Buffer Saline (PBS) pH 7.4
10 mM sodium nitropruside prepare in PBS (298mg in 100ML of PBS)
1% sulfanilamide (Sulf) (1g in 100ML of Millipore water)
2% Phosphoric acid (2.35ml in 100ml of Millipore water)
0.1% N-1-napthylethylenediamine dihydrochloride (100mg in 100ml of 2% phosphoric acid)
after this i took 1ml of plant extract (conc. 1mg/1ml) in dry test tube and then added 1ml of 10mM sodium nitropruside solution. for blank i used 1ml of 10mM sodium nitropruside solution + 1ml of methanol then all the test tubes were incubate in light at 250C for 150 minutes.
Griess reagent was prepared by mixing the equal volume of 1% sulfanilamide (Sulf) and 0.1% N-1-napthylethylenediamine dihydrochloride.
after incubation 2ml of griess reagent was added to each sample. reading were noted after 10 minutes at 546 nm.
i took sodium nitrite as standard. 1 ml of standard of different concentration + 1ml of sodium nitripruside 10mM then incubation for 150 minutes then 2ml of griess reagent.
reading are as follow
1 2 3 mean
Blank 0.176 0.187 0.216 0.193
sample 1 0.559 0.579 0.595 0.578
S-2 0.258 0.269 0.266 0.264
S-3 0.243 0.251 0.250 0.248
S-4 0.466 0.464 0.472 0.467
Now ,how to calculate nitric oxide scavenging activity?
Dear Mr. Sharma
You can calculate the % inhibition by using the following formula
% inhibition = [(A0– A1) / A0] x 100
where Ao = Abs of control, A1=Abs. of tested samples and can be compared with the absorbance of standards used.
Hope this answer your query
Neeraj K. Patel
but in this case i got negative value for sample as the absorbenc of sample was greater than control..
Dear Mr Sharma,
I think your plant sample itself contain nitrite. Thus have to maintain respective controls for your samples without any SNP addition. Then subtract this value from your sample reading.
Good luck
Dear Mr Sharma,
You can find the answer in the following attached file
Methods for measuring nitric oxide scavenging have existed for well over a decade. For example, this paper in the Journal of Medicinal Food: http://online.liebertpub.com/doi/abs/10.1089/jmf.2004.7.343# Notice the use of sodium nitroprusside as the NO donor. You might perform a literature search on one of the many search engines on the Internet to get learn of similar papers.
I have done NO. scavenging and at times with certain extracts have got negative results....that too in a dose dependent manner....we assumed that either nitrite was present in the sample or somehow NO is generated by the crude plant sample (earlier being a better calculated guess).....
NO assay itself is a tricky one where 0.33 % sulfanilamide needs to be dissolved in 20% acetic acid in deionized water (I can see that you are using only millipore water for 1% sulfanilamide)....also, I don't understand that what's the need of phosphoric acid and methanol in the test?
Its a personal experience that most of the times there is a fluctuation among the triplicate or hexaplate readings in this particular assay among all the other antioxidant assays....
I suggest you increase the molarity of SNP to 15 mM from your existing 10 mM and check....coz we use it that way n have got good results....n also that rather than using one concentration you may use multiple at least 4-5 in order to get the dose -dependent activity (if any) of your samples and in order to compare each dose effect with the control....
There is possibility of formation of nitrite from any inorgnaic source. It is possible present in the sample or somehow NO is generated by the crude plant sample, or by any other contaminant if grown in background any of the reagent. Only traces of it are required to show the alteration in absorbance.
Article Evaluation of Nitrite Radical Scavenging Properties of Selec...
please check the above link
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