Antioxidant activity is a multifaceted chemical property that is measured primarily in a lab setting using various assays. The choice of assay is critical and depends on the sample, the desired mechanism of action to study (such as hydrogen atom transfer or single electron transfer), and the available equipment. In vitro methods are the most common due to their relative simplicity, speed, and low cost.
The DPPH Radical Scavenging Assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay is a popular, rapid, and relatively inexpensive method for assessing the radical-scavenging activity of a compound. It utilizes a stable, deep violet-colored free radical, DPPH•, which turns a pale yellow color upon reduction by an antioxidant. The decrease in absorbance is measured by a spectrophotometer, providing a quantitative measure of antioxidant potential.
Procedure for DPPH Assay
- Preparation of DPPH Solution: A methanolic solution of DPPH is prepared, typically at a concentration of 0.1 mM. Since DPPH is sensitive to light, the solution and the assay should be protected from light exposure.
- Sample Preparation: The sample (e.g., a plant extract or chemical compound) is prepared at various concentrations, usually in methanol or another suitable solvent.
- Reaction Setup: Add a specific volume of the sample solution to a fixed volume of the DPPH solution in a test tube. A control tube with only the DPPH solution and solvent (no sample) is also prepared.
- Incubation: The tubes are incubated in the dark for a set period, typically 30 minutes, to allow the reaction to reach a stable state.
- Absorbance Measurement: The absorbance of each tube is measured at a specific wavelength, usually around 517 nm, using a spectrophotometer.
- Calculation: The percentage of scavenging activity is calculated by comparing the absorbance of the sample tubes to the control tube. The results are often expressed as an EC50 value, which is the concentration of antioxidant required to decrease the initial DPPH concentration by 50%. A lower EC50 indicates higher antioxidant activity.
The FRAP (Ferric Reducing Antioxidant Power) Assay
The FRAP assay measures the reducing potential of an antioxidant based on a single electron transfer (SET) mechanism. It quantifies the capacity of antioxidants in a sample to reduce a ferric iron (Fe³⁺) complex to its ferrous form (Fe²⁺), which results in the formation of an intense blue-colored product. The absorbance of this color change is measured spectrophotometrically at 593 nm. The assay is rapid, cost-effective, and widely used for assessing the antioxidant capacity of various foods and biological fluids.
Steps for FRAP Assay
- Prepare FRAP Reagent: A working solution of FRAP reagent is prepared by mixing an acetate buffer (pH 3.6), a solution of 2,4,6-tripyridyl-s-triazine (TPTZ) in HCl, and ferric chloride (FeCl₃) solution.
- Sample and Standard Preparation: Prepare the sample extracts and a standard curve using a known antioxidant, such as Trolox or ferrous sulfate.
- Reaction Mixture: Combine the sample or standard with the FRAP reagent and incubate at 37°C for a specified time, typically 30 minutes, protected from light.
- Absorbance Reading: Measure the absorbance of the mixture at 593 nm.
- Quantification: Use the standard curve to determine the FRAP value of the sample, often expressed as Trolox equivalents (TE).
The ORAC (Oxygen Radical Absorbance Capacity) Assay
The ORAC assay measures the antioxidant activity based on a hydrogen atom transfer (HAT) mechanism. It is considered more biologically relevant than some other methods because it uses a biologically relevant free radical, the peroxyl radical. The assay involves mixing the antioxidant sample with a fluorescent probe (like fluorescein) and a peroxyl radical generator (like AAPH). As the peroxyl radicals quench the fluorescence over time, the antioxidant's ability to inhibit this quenching is measured.
ORAC Assay Protocol
- Reagent Preparation: Prepare solutions of fluorescein, the peroxyl radical generator (AAPH), and the standard antioxidant (Trolox) in appropriate buffers.
- Microplate Setup: Add the fluorescein and samples/standards to a microplate. Incubate at 37°C.
- Initiate Reaction: Add the AAPH solution to each well to start the reaction.
- Fluorescence Monitoring: Measure the fluorescence intensity every minute for about 35-60 minutes using a fluorescence microplate reader.
- Data Analysis: Calculate the area under the fluorescence decay curve for each sample and the blank. The protective effect of the antioxidant is determined by comparing the net area (area of sample minus area of blank) to the net area of the standard. Results are typically expressed as Trolox equivalents (TE) per unit of sample.
Comparison of Common Antioxidant Assays
| Feature | DPPH Assay | FRAP Assay | ORAC Assay |
|---|---|---|---|
| Mechanism | Primarily Single Electron Transfer (SET) | Single Electron Transfer (SET) | Hydrogen Atom Transfer (HAT) |
| Principle | Scavenging of stable free radicals | Reduction of ferric ions to ferrous ions | Inhibition of free radical-induced fluorescence decay |
| Indicator/Probe | 2,2-diphenyl-1-picrylhydrazyl (DPPH) | Fe³⁺-TPTZ complex | Fluorescein |
| Measurement | Spectrophotometry at 517 nm | Spectrophotometry at 593 nm | Fluorimetry (fluorescence monitoring) |
| Key Advantage | Simple, fast, inexpensive | Rapid, inexpensive, simple equipment | Biologically relevant radical, measures both hydrophilic and lipophilic antioxidants |
| Key Limitation | Doesn't mimic in vivo conditions, light-sensitive | Not sensitive to all antioxidants (e.g., thiols), acidic pH | Requires specialized equipment (fluorescence reader) |
| Typical Readout | EC50 (concentration for 50% inhibition) | Trolox Equivalents (TE) or Fe²⁺ equivalents | Trolox Equivalents (TE) |
Considerations for Accurate Antioxidant Testing
- Method Selection: No single method can provide a complete picture of a sample's antioxidant capacity. It is often recommended to use multiple assays with different mechanisms (SET and HAT) to get a more comprehensive understanding.
- In Vitro vs. In Vivo: In vitro assay results do not directly translate to in vivo effects. A compound may show high antioxidant activity in a test tube but may have low bioavailability or different interactions in a living organism. Cellular-based and animal model studies are often required for a more complete picture.
- Solvent and pH: The solvent system and pH can significantly influence the results of an antioxidant assay. For example, a sample's reducing ability may be suppressed in acidic conditions.
- Interference: Pigments or other compounds in the sample can sometimes interfere with the assay's detection by absorbing light at the same wavelength as the indicator. Proper controls and sample preparation, such as deproteinization or extraction, are crucial.
Conclusion
Measuring antioxidant activity involves selecting the most appropriate assay based on the research objective. The DPPH, FRAP, and ORAC assays are three foundational methods, each with its own mechanism, strengths, and weaknesses. While in vitro tests provide a rapid and cost-effective way to screen potential antioxidants, a holistic understanding requires a multifaceted approach that considers different assay mechanisms, and ideally, includes cellular or in vivo studies. The accurate interpretation of results depends on a solid understanding of each method's principles and limitations.
Practical Applications and Future Outlook
Accurate measurement of antioxidant activity has significant applications in the food, cosmetic, and pharmaceutical industries. Researchers and manufacturers use these methods to screen for natural antioxidant sources, such as plant extracts, and to ensure product quality and stability. The ongoing development of more advanced and high-throughput techniques, including automated systems and electrochemical methods, continues to improve the efficiency and accuracy of antioxidant testing.
