Superoxide radicals are generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of NBT. In these experiments, the superoxide radicals were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 1 ml of NBT (50 mM) solution, 1 ml NADH (78 mM) solution and sample solution of fraction (25 – 500 mg/ml) in ethanol. The reaction started by adding 1 ml of PMS solution (10 mM) to the mixture. The reaction mixture was incubated at 25 °C for 5 min, the absorbance was read at 560 nm by spectrophotometer (Schimadzu UV-Vis 1700) against blank samples using ascorbic acid as a control. Decreased absorbance of the reaction mixture indicated the increasing of superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the following formula:

% inhibition = [(A0-A1)/A0] x 100

where A0 was the absorbance of the control , and A1 was the absorbance in the presence of fraction or standards.

For further details, please go through the article, "Ficus racemosa Stem Bark Extract: A Potent Antioxidant and a Probable Natural Radioprotector. Evid Based Complement Alternat Med. 2009 September; 6(3): 317–324."

Availble for download @ Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722211/

Superoxide radicals are generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of NBT. In these experiments, the superoxide radicals were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 1 ml of NBT (50 mM) solution, 1 ml NADH (78 mM) solution and sample solution of fraction (25 – 500 mg/ml) in ethanol. The reaction started by adding 1 ml of PMS solution (10 mM) to the mixture. The reaction mixture was incubated at 25 °C for 5 min, the absorbance was read at 560 nm by spectrophotometer (Schimadzu UV-Vis 1700) against blank samples using ascorbic acid as a control. Decreased absorbance of the reaction mixture indicated the increasing of superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the following formula:

% inhibition = [(A0-A1)/A0] x 100

where A0 was the absorbance of the control , and A1 was the absorbance in the presence of fraction or standards.

For further details, please go through the article, "Ficus racemosa Stem Bark Extract: A Potent Antioxidant and a Probable Natural Radioprotector. Evid Based Complement Alternat Med. 2009 September; 6(3): 317–324."

Availble for download @ Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722211/

thanks for the reply but still my question left...

please tell me , what amount i will take for control....

here no need to compare with the standard ? if yes what compound will be the standard..?

i have attached a paper here.... see the protocol and graph.....i have highlited in red colour.... here l-ascorbic acid as control...then how they have represent percentage activity of l-ascorbic acid in graph. it will not suit on this formula.....

because A0 is control

% inhibition = [(A0-A1)/A0] x 100

Dear Yogesh Kumar

Please find the link to the paper below. I also performed the same activity in my studies. You will get an idea about how to present the results of NBT assay and also get some very good references on this activity.

Please follow the link for the full text of the article:

https://www.researchgate.net/publication/215737885_Anxiolytic_activity_of_Evolvulus_alsinoides_and_Convulvulus_pluricaulis_in_rodents

Regards

Article Anxiolytic activity of Evolvulus alsinoides and Convulvulus ...

while following the protocol for superoxide ion scavenging activity what you need to keep in mind is that you are generating free radicals in presence of PMS. So when you are preparing your blank it should be without PMS because you autozero the blank while you take readings on U-V spectrophotometer. If you will add PMS to the blank you will get negative readings. The control which you have to prepare sould contain everything except your sample whose activity you are going to check. And for standard you can take any known compound with good free radical scavenging activity so that you can compare its result with your sample.

Good Luck :-)

thanks Aastha ,good information

Why blank samples should not have PMS? I don´t understand that...

.

Does this assay work at acidic pH (4.5)?

the assay requires an alkaline medium. PMS is like the catalyst that causes the generation of superoxide ion. If the blanks will serve their purpose of background reading, you do not need to add them in the blanks.

it a kinetic read that you need, then you dont need the blank ( it verry low maybe near to 0.001 )

and the control have : PMS, NBT, NADH, and have phostaphe buffer instead your simples

the right methode is like this :

To evaluate the potential to scavenge O2 radical, we creat a non-enzymatic system by the following method : phenazine methosulfate (PMS) is reduced by NADH and then reacts with oxygen, producing O2●- . Then, this radical reacts with nitroblue tetrazolium (NBT), reducing it to formazan, a blue compound which has a maximum absorption at 560 nm.

In the presence of substances capable to scavenge O2●- ( like ascorbic acide or any polyphenol ) radical, the rate of NBT reduction decreases and, consequently, it is verified a decrease in the color intensity.

so what you need to do :

mixt 50 µl of each concentration of lyophilised samples dissolved in phosphate buffer (ph 7.4 ; [19 mM]) with 50 µl of NADH [166 µM], 150 µl of NBT [43 µM] and finaly (immediately before reading) 50 µl of PMS [2.7 µM].

All essays should be conducted at room temperature, after the addition of PMS at the end, the reaction started, and it was read at 560 nm and 37 degrees C for 2 min in a 96 well plate reader.

the result of the control reading need to be between 0.2 and 0.3 :

0.2 > value of control after reading < 0.3

i added a picture of my assays for you to see the coloration that we need to have in control and it's degradation with decrease of concentration on simples.

good luck