DPPH Scavenging Assay Protocol- The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay is a widely used method to assess the ability of compounds or extracts to act as antioxidants. DPPH is a stable free radical that, when reduced, changes color from deep purple to yellow, allowing for spectrophotometric quantification of scavenging activity.
DPPH Scavenging Assay Protocol
DPPH Scavenging Assay Principle
- DPPH, a stable free radical, has an unpaired electron giving it a deep purple color and a strong absorbance around 517 nm.
- Antioxidants can donate a hydrogen atom or an electron to DPPH, neutralizing it and forming the reduced, yellow-colored DPPH-H.
- This color change is proportional to the antioxidant’s scavenging capacity. Spectrophotometric measurement at 517 nm allows quantification of the reaction.
Materials- DPPH Scavenging Assay Protocol
- DPPH (2,2-diphenyl-1-picrylhydrazyl): Purchase from a reliable chemical supplier.
- Methanol (or Ethanol): Spectrophotometric grade.
- Test Samples: Extracts, compounds, or solutions to be tested for antioxidant activity.
- Positive Control: Known antioxidant compound (e.g., ascorbic acid, Trolox).
- Spectrophotometer
- Cuvettes or Microplates: Suitable for spectrophotometer readings at 517 nm.
- Pipettes: Adjustable micropipettes for accurate volume dispensing
- Vortex Mixer (Optional): For thorough mixing of solutions.
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Safety Precautions- DPPH Scavenging Assay Protocol
- DPPH: Can be an irritant. Minimize skin exposure and inhalation. Wear gloves and potentially eye protection when handling.
- Organic Solvents: Methanol and ethanol are flammable. Handle with appropriate precautions, away from open flames and with good ventilation.
- Sample-Specific Hazards: Know the potential risks of your test samples and follow safe handling procedures.
DPPH Scavenging Assay Protocol Procedure
- DPPH Stock Solution Preparation:
- Calculate the amount of DPPH needed based on the desired working solution volume and final concentration (typically 0.1 mM).
- Weigh DPPH accurately using an analytical balance.
- Dissolve DPPH in methanol or ethanol.
- Protect from light (wrap the container in aluminum foil) due to DPPH’s light sensitivity.
- Store the DPPH stock solution in a refrigerator until use.
- Preparation of Working DPPH Solution:
- Dilute the DPPH stock solution with methanol or ethanol to achieve the desired working concentration (usually 0.1 mM).
- Check the absorbance of the working solution on a spectrophotometer at 517 nm. Ideal absorbance should be around 1.0 ± 0.1. Adjust dilution if needed.
- Prepare fresh working solution daily.
- Preparation of Test Samples and Control:
- Dissolve test samples in a suitable solvent (methanol, ethanol, DMSO, water, etc.). Ensure solubility and compatibility with the assay.
- Prepare a series of dilutions to test a range of sample concentrations.
- Positive Control: Dissolve ascorbic acid (or another chosen standard) in the same solvent. Prepare a serial dilution.
- Reaction Setup
- Add Test Sample: Pipette a defined volume of each sample dilution into separate wells or cuvettes.
- Add DPPH: Add an equal volume of DPPH working solution to each well/cuvette (this initiates the reaction).
- Positive Control: Repeat with dilutions of ascorbic acid or your chosen standard.
- Negative Control (Blank): Include a reaction containing only solvent and DPPH solution.
- Incubation
- Mix solutions thoroughly (pipetting up/down or gentle vortexing).
- Incubate in the dark at room temperature for a defined time (often 30 minutes). Incubation time optimization may be needed.
- Absorbance Measurement
- After incubation, measure the absorbance of each reaction mixture at 517 nm on the spectrophotometer.
- Use the blank (solvent + DPPH) to zero the instrument.
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Table-1: DPPH Scavenging Assay protocol
| Methodology Step | Description | Important Considerations |
|---|---|---|
| DPPH Preparation | Prepare a DPPH stock solution in methanol or ethanol. Protect from light. | Use a spectrophotometric-grade solvent. DPPH is light-sensitive. |
| Working Solution | Dilute the stock to the desired concentration (often 0.1 mM). Check absorbance at 517nm. | Freshly prepare the working solution daily. |
| Sample & Control | Prepare samples and a positive control (e.g., ascorbic acid) at various dilutions in a suitable solvent. | Ensure good sample solubility. Choose a solvent compatible with the assay. |
| Reaction Setup | Combine samples and controls with an equal volume of DPPH working solution. Include a solvent-only blank. | Mix thoroughly to initiate the reaction. |
| Incubation | Incubate in the dark for a set time (e.g., 30 minutes). | Choose incubation time based on the kinetics of your antioxidant. |
| Measurement | Measure the absorbance of each reaction at 517 nm using a spectrophotometer. | Zero the spectrophotometer with the blank. |
| Data Analysis | Calculate % scavenging activity for each sample. Determine IC50 values. | Correct for any significant background absorbance of the samples. |
Data Analysis and Calculations
Scavenging Activity = [(Abs control – Abs sample) / Abs control] x 100
Where: * Abs control = Absorbance of the negative control (DPPH + solvent) * Abs sample = Absorbance of the sample reaction mixture
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IC50 Calculation
- Plot the % scavenging activity vs. sample concentration (or log concentration for a wider range).
- Determine the sample concentration that causes 50% reduction of DPPH (the IC50 value). This represents the antioxidant’s potency. A lower IC50 indicates greater antioxidant activity.
Interpretation of Results
- Comparison to Positive Control: Evaluate the test sample’s scavenging capacity relative to the known antioxidant standard.
- Structure-Activity Relationships: If analyzing a series of related compounds, results can aid in investigating the impact of molecular features on antioxidant activity.
Additional Considerations
- Solvent Choice: The solvent used for both DPPH and your samples can influence solubility and antioxidant activity. Methanol and ethanol are most common, but adjustments might be needed.
- Kinetics: The DPPH reaction rate varies with different antioxidants. Ensure the chosen incubation time is sufficient, or consider time-course studies.
- Interferences: Some compounds may absorb at 517 nm, interfering with the assay. Include sample-only controls (without DPPH) to correct for background absorbance.
- Alternative Detection: While absorbance measurement is standard, methods like HPLC or electron paramagnetic resonance (EPR) can be used in more complex cases or for structural analysis of the products.
Troubleshooting
- Low Scavenging Activity:
- Increase sample concentrations
- Extend the incubation time.
- Ensure sample solubility and compatibility with the solvent.
- Inconsistent Results:
- Verify pipetting accuracy and calculations.
- Check the stability and freshness of DPPH solution.
- Control for temperature variations if they significantly impact your samples.
- Unusual Absorbance Readings:
- Check for precipitation in the reaction mixtures.
- Exclude interference by sample or solvent by measuring their absorbance individually.
Advantages of DPPH Assay
- Simple and Rapid: Easy to perform, with relatively quick results.
- Versatile: Can be applied to various types of antioxidants and samples.
- Cost-Effective: Requires basic laboratory equipment and reagents.
Limitations of DPPH Assay
- Not Biologically Predictive: Antioxidant activity in vitro doesn’t always translate directly to activity within a living system.
- Limited Specificity: DPPH is a synthetic radical; results shouldn’t be over-extrapolated to infer scavenging of all types of reactive oxygen species (ROS) found in biological systems.
Contact For DPPH Scavenging Assay
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