I am trying to study antioxidant activity of some plant extract , for that i am doing hydroxyl radical scavenging activity study but even if i have done so many method with different concentration of reagents and Gallic acid as a standard i am unable to get it.
i have followed so many articles but still i am not getting the result of standard
here is the reagent i am using
Standard gallic acid
FeCl3 10 mM
EDTA 1.04mM
2-deoxy ribose 28mM
H2O2 10mM
Ascorbic acid 1mM
TBA 1% and TCA 10%
for antioxidant activity you need:
1-sample (2ul)
2- DMSO (2ul)
3-Vit C (2ul) ( 5 mmol)
4-methanol (50 ul)
5- DPPH ( 50ul)
Hydroxyl radical reacts with almost all organic molecules with a diffusion controlled rate constant. None of antioxidant can protect any organic molecules from damages caused by hydroxyl radicals.
In addition, different radicals (but not hydroxyl) will be generated in your system.
Evaluation of antioxidant activity requires a very deep understanding of chemical kinetics and chemistry of free radicals.
Read reviews, choose one of the standard protocol and strictly follow it. Don't waste your time inventing your own protocol.
Hi Geletii Sir ,
thanks for responding my queries, i have tried so many standard protocol still i am not getting it as the std/extract concentration increases the color intensity also increase instead of decreasing, if you suggest me any standard protocol that will be very helpful for me.
Thanks.
The Briggs-Rauscher reaction can be used as a test to measure the activity of antioxidants. See the attached paper.
Hi
greeting for the Day Schmitz sir. thanks for the paper, I will go through the paper hope it will help me.
Reducing power assay
In sample extract (1mg/mL) at different concentrations , add 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide . Incubate for 20 min at 50°C then add 2.5 mL of 10% trichloro acetic acid and centrifuge at 3000 rpm for 10 min. 2.5 mL of supernatant was added to 2.5 mL distilled water and 0.5 mL 0.1% ferric chloride. Read absorbance at 700nm against a blank. Ascorbic acid - positive control
Hi
Deepika thanks for your suggestion could you please mail me the reference paper then it will be easy for me to understand the mechanism of the reaction. and yap i ll go through this protocol hope it will solve my problem.
thanks
For Hydroxyl Radical Scavenging Activity - Hydroxyl radical generated by Fe+3–Ascorbic acid–EDTA–H2O2 system (Fenton reaction)
The reaction mixture (1.0 ml) of
0.1 ml 2-deoxy-2-D-ribose (28 mM in 20 mM KH2PO4–KOH buffer, pH 7.4),
0.5 ml of the different concentrations (μg/ml) of sample,
0.2 ml EDTA (1.04 mM) and 200 μM FeCl3 (1:1 v/v),
0.1 ml H2O2 (1.0 mM), and
0.1 ml ascorbic acid (1.0 mM)
Incubation at 37 °C for 1 h.
1.0 ml TBA (1%) and
1.0 ml TCA (2.8%)
Incubated at 100 °C for 20 min.
OD at 532 nm against a blank sample...
Here I attached a review paper on antioxidant assays. It can be useful as well.
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