You need to apply the same method described on your previous question on Gallic Acid ... That just mean that you are considering all reaction on your colorimetric quantifiation as a Gallic/Ascorbic reaction. I think you can find thoses kind of answer on google.

I have done all that Adrien. I just cant lay my hands on an appropriate conversion equation or formula. Most journals and search on google just use the statement: Results were expressed in Gallic/Ascorbic acid equivalent/100g. i would really appreciate that conversion phase.

DPPH assay

Each analysed extract (0.15 mL) was mixed with 2.85 mL freshly prepared 0.1 mM solution of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH, Sigma) in methanol. The reaction was performed at 37 °C in darkness and the absorbance at 517 nm was recorded after 15 min against methanol. The antioxidant activity was expressed as mM Ascorbic acid equivalents (TE) per g dry weight (DW) by using calibration curve, build by 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mM ascorbic acid dissolved in methanol.

 Dear Johnson Esua,

First of all you have to draw a standard curve with different concentration of ascorbic acid and find out the straight line equation of that curve (eg. y=mx+c). In the equation you will get a value for m and also for c. Ignore the value c and consider the equation as y=mx. Now put your OD value of unknown concentration in place of y and equate thereafter. 

FOR EXAMPLE- If I have 200 mg of sample and my OD value of DPPH is 0.617 and my total reaction mixture (RM) is 10 ml and the straight line equation for standard curve is y=0.008x+0.0429. then,

0.617=0.008x

x=617/8=77.125

therefore, 10 ML RM contains 77.125 µg/ml or 771.25 µg.

again, 200 mg or 0.2 gm of sample contains 771.25 µg or 0.77125mg

therefore, 100 gm of sample contains 385 mg AAE (ASCORBIC ACID EQUIVALENT)/100 gm dry weight.

THANKING YOU,

SANDIPAN RAY (UGC-SRF)

Thank you Sandipan Ray. Just a little clarification. What do you mean by OD value of DPPH? Is it the percentage (%) scavenging activity of DPPH obtained for the sample? By Total Reaction Mixture (RM) are you referring to sample extract + DPPH solution or Ascorbic acid+DPPH solution?

Very simple: Use standard pure ascorbic acid for your DPPH test and plot a standard curve and use it to measure AA/100g of your sample.

 OD value means OPTICAL DENSITY VALUE READING which you got from spectrophotometer. Reaction mixture is the total volume of ur sample or standard with DPPH.

Sorry Ray. I still need more clarifications. You mean OPTICAL DENSITY VALUE READING is the absorbance reading which I got from spectrophotometer for the extract. What about other parameters like the absorbance of the control? I used these two readings to obtain the % scavenging activity from the formula

Scavenging effect (%) =[1- (OD sample/ OD control)] * 100.

Are you saying I do not need to calculate this scavenging effect, but I should use the OD directly in your equation?

Dear Johnson, once you get your sample reading, just use the calibration curve drawn using ascorbic acid and calculate the concentration of your sample extract in equivalent units that was used for the ascorbic acid. Briefly, if the unit used when you prepare ascorbic acid standard was mg/ml, when you insert the absorbance value of your sample in to the calibration equation, the concentration of your sample will be in units of mg/ml equivalent to ascorbic acid and multiply this by the total volume of the extract. The final value obtained will be the antioxidant capacity of your sample extract (i.e. of the amount of sample you used during extraction) and then you can calculate the amount to be obtained from 100g sample and report your result in mg/100g sample.

Johnson, you do't need to calculate % scavenging effect if you want to express the result in AAE, that one can indipendetly be used to report the result.

Thank you so much Endale. You have solved all my problems. Does the Total volume of extract refer to the volume of extract I combined with reagents to get an absorbance, or the total extract obtained from the extraction initially?

idem Fatima

WHY not express your results in  IC50 (mg /mL) or Total antioxidant  equivalent Capacity (TAEC) which normally or conventionally used.

by utillise IC50 in mg/mL

Dear Johnson, total volume of extract refers to the later you mentioned (i.e. the total extraction volume you have during extraction).

Thanks Endale. You were really helpful.

Thanks Endale. You were really helpful.

is better express the results in IC50 (mg /mL) in conventionally used.

You have to express Ascorbic acid equivalent as IC50 value of your graph.

I have a query @ Sandipan Ray. When the scavenging potential increases the absorbance at 517 nm keeps on decreasing. If we use the same absorbance for calculation of AA, it would obviosuly come out low. But actually with increase in scavening potential, AA should increase. How can we then directly use the absorbance for AA calculation.

What you have observed is perfectly right. Absorbance will decrease with increasing scavenging potential. AA will decrease with increase in scavenging potential. To calculate the AA concentration it will always be a function of time.

The point I want to make is that if m comparing 5 different samples and say from 1 to 5 the scavenging potential increases, then how m I going to calculate AA on comparative ground for them

Then the (Abs-control - Abs-sample) should be used for y=mx+c. Isnt it', rather than the Abs for each sample.

Well....., you may do so.

But the point is I lack a supporting document for this. that

See if the attached reference would be of some help?

Thanks, Jai Ghosh for added pdf very resourceful.

Agree with Dr Jai Ghosh