DPPH radical scavenging assay via micrototre plate method.pptx

the ppt describes the DPPH RADICAL SCAVENGING ASSAY using 96- well microtitre plate

1. ANTIOXIDANT ACTIVITY: DPPH Radical Scavenging Assay (Microtiter Plate Method) By: RIPPIN

2. Free Radicals Group of atoms with one or more unpaired electrons in outer orbital Highly unstable and very reactive species Types : ROS, RNS, metabolic intermediates (metabolic activation of drugs, toxins, pollutants etc ). Examples : superoxide anion radical, hydrogen peroxide, hydroxyl radical, nitric oxide radical etc

3. Harmful effects of Free Radicals React with biomolecules Enzymes Nucleic acids Carbohydrates Cellular Disturbances Tissue Injury DISEASES

4. ANTIOXIDANTS Interact with and stabilize FREE RADICALS Prevent the cells from damage Prevent the development of various diseases

5. Determination of Anti- oxidative potential of Antioxidants in samples (Plant Extracts) DPPH ( 2,2-Diphenyl-1-picrylhydrazyl) Assay Diphenyl picryl hydrazine

6. D ouble Dilution Method (de torre et. a l. 2019) Reagents: 0.1 mM DPPH, Methanol, 10mg/ml Ascorbic acid(positive control), 100µl DPPH+100µl methanol(control), 10mg/ml sample antioxidant 100 µl methanol in each well 10 µl of extract in 1 st well (500µg/ml) + 90 µl Methanol Transfer 100 µl of mix from 1 st to next well and so on Discard 100 µl from the last well 100 µl DPPH in each well The final conc in each well will become half Repeat for positive ctrl Add control in a separate well Cover the plate with foil and keep in dark fro 30 mints Read at 517 nm via microplate reader Ctrl DPPH+Methanol

7. Results 250 µg/ml 15.64µg/ml 7.8 µg/ml 3.9µg/ml 1.95µg/ml 125 µg/ml 62.5 µg/ml 31.25µg/ml Scavenging effect (%) = {(Absorbance of control− Absorbance of sample)/Absorbance of control} × 100

8. IC 50 Value IC 50 (half maximal inhibitory concentration) the concentration of the antioxidant which reduces DPPH to half i.e. 50 %. Lower the IC 50 value, higher is the antioxidant capacity of the antioxidant.

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