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Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices

Introduction

Reactive oxygen species are generated by normal cellular processes, environmental stresses, and UV irradiation. These include peroxyl radicals (ROO.), hydroxyl radicals (HO.), the superoxide ion (O2-.) and the singlet oxygen radical (¹O₂). The oxygen radical absorbance capacity (ORAC) assay is a standard method used to assess antioxidant capacity of physiological fluids, foods, beverages, and natural products. The assay quantitatively measures a sample’s ability to quench free radicals that have the potential to react with and damage cellular components, causing mutations that can adversely affect the cell cycle and cause cancer or other diseases.

This application note describes the use of SpectraMax® Gemini^™ XPS Microplate Reader and SoftMax® Pro Software with Cell Biolabs OxiSelect^™ ORAC Assay to determine the antioxidant capacity of different fruit juices and highlight the simplicity of this assay.

Assay principle

This assay is based upon the effect of peroxyl radicals (ROO.) generated from the thermal decomposition of 2, 2’-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) on the signal intensity from the fluorescent probe, fluorescein, in the presence of an oxygen radical absorbing substance. The stronger the absorbing capacity, the more the peroxyl radicals are quenched, thus maintaining the intensity of the fluorescent signal observed. In order to obtain a result, the area under the curve of the fluorescence intensity versus time is subtracted from that of the blank to determine the antioxidant capacity of the substance present, in Trolox^™ equivalents, a vitamin E analogue used as the standard measure of antioxidant capacity.

Materials

The following materials were used to perform the assay:

• Cell Biolabs OxiSelect ORAC Assay, cat. #STA-345
• SpectraMax Gemini XPS Microplate Reader
• Solid black Greiner FLUOTRAC ^™ 200 plate
• Samples: white cranberry juice, white grape juice, orange juice, pomegranate juice, cranberry-blueberry juice, and red wine

Methods

The assay setup procedure outlined below is based upon the Cell Biolabs OxiSelect Oxygen Radical Antioxidant Capacity (ORAC) Activity assay procedure provided with the kit.

Preparation of reagents

Fluorescein probe preparation: The Fluorescein probe provided was diluted 1:100 with the assay diluent provided. This was then mixed to homogeneity and labeled as the 1X Fluorescein solution.

Assay diluent preparation: The assay diluent was diluted 1:4 with deionized water and mixed to homogeneity. This was used for all sample and standard dilutions and stored at 4°C.

Sample preparation: Juice and wine samples were centrifuged at 5000–10,000 x g for 10 minutes at 4°C to remove any particulates. All supernatants were diluted 100- and 1000-fold prior to running the assay.

Preparation of antioxidant standard curve

Standards were prepared by diluting the 1000 μM antioxidant standard stock solution in assay diluent to obtain the final desired concentrations. Only enough antioxidant standard was prepared as required for immediate use. A series of antioxidant standards was created according to Table 1 below. The zero Trolox standard was treated as the blank.

Table 1. Preparation of standards

Note: Each antioxidant standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

Calculation of results

Step 1. Using SoftMax Pro Software, the area under the curve (AUC) was calculated for each sample and standard.

Step 2. The net AUC was calculated by subtracting the blank AUC from the AUC for each sample and standard.

Step 3. The net AUC values were then plotted on the y-axis versus the Trolox antioxidant standard concentration on the x-axis.

Step 4. The μM Trolox Equivalents (TE) for unknown samples were then calculated based on the standard curve, with the results expressed as μmol TE per mL of sample.

Results

SoftMax Pro Software was used for standard curve graphing and all data analysis. Kinetic curves for Trolox standards are shown in Figure 1. Area under the curve (AUC) was calculated for the blank, and this value was subtracted from each standard to calculate net AUC. Net AUC values for Trolox standards were plotted against concentration to obtain the standard curve in Figure 2.

Figure 1. Kinetic curves for different Trolox concentrations. Representative kinetic curves for Trolox standards ranging from 2.5 to 50 μM are shown. Kinetic curves are used to calculate area under the curve (AUC), which is the basis for the calculation of ORAC values.

Figure 2. Trolox standard curve. The Trolox standard curve was generated by calculating net AUC for each standard as described above (r² = 0.996).

Net AUC values were calculated for all samples. ORAC values, expressed as Trolox equivalents (TE), were back calculated from the Trolox standard curve and multiplied by the sample dilution factor to obtain final ORAC values, expressed in μmol TE/mL. Samples diluted 1:100 fell out of range of the standard curve, but samples diluted 1:1000 were all in range. ORAC values for the 1:1000 diluted samples are shown in Table 2.

Table 2. ORAC values for fruit juice and wine samples.

Among the samples tested, red wine had the highest ORAC value, and white cranberry juice had the lowest. Note that values obtained using samples from different manufacturers are likely to differ, as ingredients may vary.

Conclusion

Cell Biolabs OxiSelect Oxygen ORAC Activity Assay can be easily performed using the SpectraMax Gemini XPS Microplate Reader. Graphing of standards and calculation of results are performed automatically by SoftMax Pro Software, which is provided with the reader.

Please note that the SpectraMax M-series, i3x, iD3/iD5, and Paradigm® Multi- Mode Microplate Readers, as well as the FilterMax^™ F5 Multi-Mode Microplate Reader, all offer fluorescence detection and can also be used with the OxiSelect ORAC assay. The FlexStation® 3 Multi-Mode Microplate Reader also offers on-board pipetting for automated reagent addition to assay plates.

References

Huang, D., Ou, B., and Prior, R.L. (2005). The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 53, 1841–1856.

Wang, H., Cao, G., and Prior, R.L. (1996). Total antioxidant capacity of fruits. J. Agric. Food Chem. 44, 701–705.

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