Is there any simple procedure or SOP to measure the antioxidant activity. Please help me. Is it necessary to draw the calibration curve by the standard sample such as Vitamin C or E. I saw some procedure to measure activity against the blank but I am confuging for this procedure.
Use the attached method but use the below standard curve protocol.
Firstly make ascorbic acid stock 1 (10mM) : Add 0.0176g Ascorbic Acid Powder to 10ml methanol or ethanol (ethanol is safer as it's a lot less toxic) or water or ethanol.
Make stock 2 (5mM): add 500 microliters stock 1 to 500 microliters water or ethanol.
Make stock 3 (250uM): Add 50 microliters stock 2 to 950 microliters water or ethanol.
Use stock 3 to make standard curve.
Standard curve
Ascorbic Acid Concentration (uM) microliters stock 3 microliters water
0 0 100
5 2 98
10 4 96
25 10 90
50 20 80
125 50 50
250 100 0
This should give you an excellent standard curve.
the simplest one is to calculate the percentage scavenging which can be calculated using (control-sample)/control x 100. here, control is without your sample (only the solvent you used for dissolving your extract and of course DPPH)..... another method is drawing a standard curve using standard antioxidant (trolox, vit E etc) and express as your standard equivalent....... in both the cases, the procedure is same, just the expression is different...
I agree with Lalrinzuali Khawlhring's answer, Find IC50 values after calculating percentage scavenging activity at different concentrations.
Use the attached method but use the below standard curve protocol.
Firstly make ascorbic acid stock 1 (10mM) : Add 0.0176g Ascorbic Acid Powder to 10ml methanol or ethanol (ethanol is safer as it's a lot less toxic) or water or ethanol.
Make stock 2 (5mM): add 500 microliters stock 1 to 500 microliters water or ethanol.
Make stock 3 (250uM): Add 50 microliters stock 2 to 950 microliters water or ethanol.
Use stock 3 to make standard curve.
Standard curve
Ascorbic Acid Concentration (uM) microliters stock 3 microliters water
0 0 100
5 2 98
10 4 96
25 10 90
50 20 80
125 50 50
250 100 0
This should give you an excellent standard curve.
Hi Yunus, if you have the possibility of using ESR spectroscopy at your workplace, it is a very easy and fast tool for determination. If you need more information, tell me. Best regards.
we can used this simple method:
DPPH radical scavenging activity
The radical scavenging activity using free-radical DPPH assay was carried out using this method. 1 mL aliquot of each extract was added to 0.5 mL of a DPPH ethanolic solution (7.8 mg DPPH in 100 mL ethanol 70 %). The mixture was vigorously shaken and left to stand in the dark for 30 min at room temperature. The antioxidant activity was then measured by the decrease in absorption at 517 nm using UV-Visible spectrophotometer and corresponds to the extract ability to reduce the radical DPPH to the yellow-colored Diphenylyldrazine. The Anti-radical activity was expressed as IC50 (μL/mL) i.e. the Anti-radical percentage inhibition calculated by the following equation:
DPPH Inhibition % =[ (Ao ̶ A1)/Ao] x100
Where Ao is the absorbance of control test after 30 min. A1 is the absorbance of the sample extract after 30 min. All results are means ±SD
Can these DPPH activity protocols be used for n-hexane, chloroform, ethyl acetate plant extracts?
Ashwini you may need to dissolve the non-polar extracts in non-polar solvents and visa versa.
Hi Naim
usually 0 concentration of the standard as you mentioned, has zero absorbance or some abs value may or may not be added in calibration curve?
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