I referred several article based on DPPH assay but each article used different concentration of DPPH and different volume ratio. According to my knowledge, concentration of DPPH won't be a problem as we will substract the absorbance at the end but when we change the volume ratio of the ascorbic acid and DPPH, the IC50 value will change due to the scavenging capacity.
For example,
when I used 200 microliter extract and 2.8 ml DPPH, IC50 value around 40 tp 50 ppm while when I used 100 microliter extract and 3.9 ml DPPH, IC50 value around 130 to 140 ppm.
Both values are acceptable or is there any specific value or procedure to follow?
In DPPH scavenging assay the IC50 value of the extract was found to be 43.26µg/ml while the IC50 value of the reference standard ascorbic acid was 58.92 µg/ml. https://www.banglajol.info/index.php/BJVM/article/view/2336/2051
Also check https://www.scielo.br/pdf/bjps/v54n1/2175-9790-bjps-54-01-e17178.pdf
Thank you very much Chinaza Godswill Awuchi Sir. I have confusion in the volume ratio and the IC50 values. because when we change the volume ratio, IC50 value also changing. different papers have different IC50 values for ascorbic acid due to the difference in the volume ratio maybe. but when I converted all values into same procedure, all values are approximately same. That means, depending on the procedure, the value can be changed. is it the fact?
Thanks in advance
There is no sense to compare the data collected under different conditions. You have to calibrate your assay under your conditions with respect to equivalents of ascorbic acid. In this case IC50 becomes relative unitless value.
I really appreciate Yurii V Geletii Sir. Thank you very much. your answer really helpful to me.
DPPH assay would give the best result under conditions:
1. using methanol buffer
2. final concentration of DPPH in the reaction is 25 - 70 µM (absorbance 0.221-0.698)
3. the assay performed in a dark ambiance (± 30 ◦C).
the IC50 of ascorbic acid would be ± 2 µg/ml
Kokilananthan Shanthirasekaram Hi...i see your question and i want to ask why you choose this values? IC50?
Roxana Nezami hello... Actually, I did not choose any IC50 value for ascorbic acid but according to my knowledge gain after several trial with different DPPH assay, the IC50 value for ascorbic acid or any plant samples will be vary with your procedure. Specially, with the volume ratio of the sample/standard and DPPH solution. As I am still not satisfy with this statement, I am trying to inter correlate with each procedure to find out is there any relationship between volume ratio and IC50 value. Please correct me if I am wrong
Kokilananthan Shanthirasekaram That's right...thank you for your response
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