Hi Eyop,

For helping you, you have to provide some information:

- What's the TAC method you used in the determination of Antioxidant activity?

- What's the type of your sample?

- What's the standard molecule you used in your method ?

- What did you mean by "control"?

The formula you cited indicates that you maybe measured the scavenging activity (%) of a stable free radical such as DPPH or ABTS. If it's the case, your control must be the standard molecule before adding the sample. In this case, the absorbance value of control must be higher and it must decrease after adding the sample due the reaction of antioxidants found in sample with a part of standard molecule.

I agree with Duried that the question is ill formulated and therefore can't be answered.

Eyob Chukalo Chutulo, before using whatever technique you should learn how it works, what is the chemistry/physics behind the technique. The most likely that a product of oxidation of your sample is colored and you measure the formation of this product. If possible. please, provide the spectrum of you sample after the reaction with ABTS-radical.

First, you have to make a blank for your samples, so you will eliminate the absorbance of your extract. Thus, it is necessary to make a dilution for the samples whose absorbance and greater than 1.

To Duried Alwazeer:

-The assay method I used is Phosphomolybdate assay method

-the sample is crude extract

- The standard is Ascorbic acid=control

-Absorbance was recorded at 695 nm

Phosphomolybdate assay method is the worst assay to measure TAC. It's not performed under physiological conditions, e.g. too high acidity. The answer to your question is simple: use 4-times higher ascorbic acid solution for the control

Yurii V Geletii

Thank you for answering my question. You mean I should use 4-times higher ascorbic acid concentration for control than the sample?

I agree with Yurii V Geletii to use more concentrated Ascorbic acid. If it doesn't work, try to use a more diluted sample with a dilution factor of 4 for example.

Actually, you need a calibration plot "absorbance versus [ascorbic acid]" to determine TAC in ascorbic acid equivalents. The better would be to measure TAC in Trolox equivalents. Again, phosphomolybdate assay method is not good at all, I do not recommend it. Use ABTS method.

Again I agree with Yurii V Geletii for the use of Trolox instead of Ascorbic acid due to the low stability of AA. The alternative TAC methods as Yurii said are ABTS or DPPH with using Trolox as a standard (control).

Duried Alwazeer

Thank you for your suggestion and reasoning. I will use it.

Yurii V Geletii

Many thanks for your concerned comments and suggestion. Yeah, I have done the calibration plot as well. Actually, I have used DPPH and reducing power assay. I wanted to compare the different methods.

My doubt was I see some literature reporting the percent radical scavenging activity by Phosphomolybate assay method; which is common to DPPH, ABTS and other radical species; instead of reporting as ascorbic acid/ Trolox equivalents /g of the extract.

In my case, the percent radical scavenging capacity calculation gives a negative result, which would not work. As a result, I report as standard antioxidant equivalents. Just to clear the doubts!.

Thank you very much!

You got a negative value because your plant extract has a high TAC than your solution of ascorbic acid. As I advised you increase the concentration of ascorbic acid in your "sample"

Yurii V Geletii

Thank you very much. I will do accordingly.

Eyob Chukalo Chutulo

What was the composition of your control (the one you obtained Abs = 1.25) ?

Kind regards

Hey, did you calculated IC50 using the standard equivalents from the standard graph?

Phosphomolybdate assay (total antioxidant capacity) The total antioxidant capacity assay of samples was carried out by the phosphomolybdenum method . A 0.1-ml aliquot of the sample solution was shaken with 1 mL of reagent solution (0.6M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The test tubes were covered and incubated in a water bath at 95 °C for 90 min. After the samples were cooled, the absorbance of the mixture was measured at 765 nm. Ascorbic acid was used as standard. The antioxidant capacity was estimated using the following formula:

Total antioxidant capacity (%) = [(Abs. of control − Abs. of sample)/(Abs. of control] × 100