Application of manual assessment of oxygen radical absorbent capacity (ORAC) for use in high throughput assay of "total" antioxidant activity of drugs and natural products.

INTRODUCTION

Antioxidants are of particular interest in a spectrum of diseases, and thus are an active area of drug discovery and design. It is important to make considered choices as to which assay chemistry will best serve for particular investigations. We examined the manual oxygen radical absorbent capacity (ORAC) assay for "total" antioxidant activity, including a direct comparison to an alternative technique, the AOP-490 assay, using a panel of extracts from 12 phylogenetically unrelated algae.

METHODS

The AOP-490 assay was done per manufacturer's protocol. The ORAC assay was done by hand, in 96-well plates, not by machine as had been previously published. Our ORAC calculations were done using an in-experiment antioxidant standard curve. Results were reported as equivalents of the antioxidant Trolox, which was used as a standard.

RESULTS

With the AOP-490 kit (from Oxis Research) widespread activity was found, but not in all samples. When the ORAC method was used to assay aliquots of the same extracts there was significant activity detected in all samples, and the rank order of activity by the two methods was not identical. The data showed the wide occurrence of antioxidants in algae. The standard curve with the manual ORAC assay was linear in the range tested (0-100 mM Trolox) and had excellent reproducibility.

DISCUSSION

The importance of the beneficial effects of antioxidants is currently an area of active interest for drug development, and thus it is of great value to have an assay that is robust and approximates "total" antioxidant activity in a high throughput format. The ORAC (oxygen radical absorbent capacity) method was adapted to microplates and an eight-channel pipette and was more effective in detecting "total" antioxidant activity than the AOP-490 assay. These results might vary with other types of samples, and would depend on the active agents measured, but do suggest the practical value of the ORAC assay for any laboratory not ready for robotics but using manual 96-well format assays, and the utility of the ORAC assay for evaluating algal, and probably other samples as well.