Dear all
what the best solvent to dissolve DPPH? before that I used 70% ethanol as file attached, but it not fully dissolved. Besides that I quite confused, what the important is IC50? why should looking for IC50. The second question which one is the better way to detect antioxidant capacity, either use DPPH or trolox?
Regards
Azlan
Hi Azlan, I use ethanol to dissolve DPPH as it's alot safer than methanol. I add 0.0238g DPPH to ethanol and make up to a final volume of 100ml. This is your stock (0.6mM). Store in an amber bottle or cover the flask with aluminium foil to protect from the light and store in the refrigerator. For your DPPH/MetOH working solution (0.06mM) add 10mL DPPH stock to 90mL ethanol (1/10 dilution).
IC50 tells you how much of the sample is required to reduce DPPH by 50% which tells you how potent your sample is. It also allows you compare results from other studies alot easier.
As for Trolox I normally use it as an antioxidant control although I prefer ascorbic acid.
Thanks replying my question. let say concentration sample (0.2,0.4,0.6,0.8,1,1.2)mg/ml. Can I use equation 3.1 to calculate % of radical scavenging activity started 10-100%. The picture and formula as attached for your perusal. After plot the graph as Figure 2, to know IC50, i just see IC50 directly on the graph. Am I right?
First make a curve of your samples. Make the following dilutions: 1 in 50 , 1 in 25 , 1 in 10, 1 in 5 and 1 in 2 and find the abs. In my case the dilutions worked out at 0.02,
0.04, 0.1, 0.2 and 0.5%.
Make your curve and get the equation of the line. Lets say the DPPH control is 0.651 then divide by 2 to give 50% pf it activity= 0.3255. Lets say the equation of the line is y = -0.5168x + 0.6085=> ((0.3255-0.6085)/-0.5168)= 0.55% sample required to reduce DPPH by 50%.
Nice explanation. Thanks. May i know, let say if we wanna check Total phenolic content, we will use gallic acid as standard curve, so for antioxidant activity, can i use trolox as standard curve instead of IC50? In this case my sample is pitaya peel extraction, based on what you have mentioned, I have to make series dilution of pitaya peel extraction(e.g:0.02-0.5%) to find the Abs and make a curve. Am I right?
When we use gallic acid as reference the results are expressed as Gallic Acid Equivalents GAE similarly for antioxidant activity we use a reference to compare. Ascorbic acid is also used as reference. IC50 indicates the potency of the compound or extract in absolute value.
For TPC assay gallic acid or phloroglucinol is normally used. You can use Trolox to give Trolox equivalents in DPPH assay. Yes you would make series dilution of pitaya peel extraction to find the Abs and make a curve. The figures I mentioned (0.02-0.5%) are only what I used so don't use that as an absolute guide. Instead you need to see how potent your extracts are, then you can decide on the appropriate dilutions.
DPPH radical scavenging activity
The free radical scavenging activity of methanol extract was measured by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) using the method of Blois (1958). 0.1 mM solution of DPPH in methanol was prepared and I ml of this solution was added to 3 ml of various concentrations of methanol extract and reference compound (125, 250, 500 and 1000 µg/ml). After 30min, absorbance was measured at 517 nm. BHT was used as a reference material. All the tests were performed in triplicate and the graph was plotted with mean value. The percentage of inhibition was calculated by comparing the absorbance values of reference compound (control) and samples.
% of inhibition = [(A blank/A sample)/A blank] x 100
Where ‘A blank’ is the absorbance of the control reaction (containing all reagents except the test sample) and ‘A sample’ is the absorbance of the extracts/standard.
Blois, M.S. 1958. Antioxidant determination by the use of a stable free radical. Nat. 181: 1199-1200.
Hi
Please use 100% methanol or ethanol, but I preferred Methanol. Anyway if you dissolved in methanol or ethanol, it takes about 30 mins to dissolved well.
Regards
Ali
Thanks A.M Naim...Your comment is so helpful...I wanted to prepare 0.6mM stock solution..
hello, i prepared 0.6mM of DPPH in methanol and vortex it with ascorbic acid for 30 minutes in dark.
now i need a reference to compare the IC50 value for this condition.
I have made 0.6mM stock solution of DPPH and used this for my assay. What is the absorbance of the control sample without addition of ascorbic acid or plant extract.
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