Price Joseph A, Sanny Charles G, Shevlin Dennis
Department of Pathology, OSU-COM, 1111 W. 17th. St., Tulsa, OK 74107, USA.
J Pharmacol Toxicol Methods. 2006 Jul-Aug;54(1):56-61. doi: 10.1016/j.vascn.2005.11.002. Epub 2005 Dec 5.
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.
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.
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.
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.
抗氧化剂在一系列疾病中备受关注，因此是药物发现和设计的一个活跃领域。对于特定研究而言，选择最适合的检测化学方法至关重要。我们研究了用于检测“总”抗氧化活性的手动氧自由基吸收能力（ORAC）测定法，包括与另一种技术AOP - 490测定法进行直接比较，使用了一组来自12种系统发育无关藻类的提取物。
AOP - 490测定法按照制造商的方案进行。ORAC测定法是手工在96孔板中进行的，而不是像之前发表的那样通过机器进行。我们的ORAC计算使用实验中的抗氧化剂标准曲线。结果以抗氧化剂Trolox的等效物形式报告，Trolox用作标准。
使用AOP - 490试剂盒（来自Oxis Research）发现了广泛的活性，但并非在所有样品中都有。当使用ORAC方法检测相同提取物的等分试样时，在所有样品中都检测到了显著活性，并且两种方法的活性排名顺序并不相同。数据表明藻类中广泛存在抗氧化剂。手动ORAC测定法得到的标准曲线在所测试的范围内（0 - 100 mM Trolox）呈线性，并且具有出色的重现性。
抗氧化剂有益作用的重要性目前是药物开发中一个活跃的研究领域，因此拥有一种稳健且能以高通量形式近似“总”抗氧化活性的检测方法具有很大价值。ORAC（氧自由基吸收能力）方法适用于微孔板和八通道移液器，并且在检测“总”抗氧化活性方面比AOP - 490测定法更有效。这些结果可能因其他类型的样品而异，并且取决于所测量的活性剂，但确实表明了ORAC测定法对于任何尚未准备好使用机器人技术但使用手动96孔板形式测定的实验室的实用价值，以及ORAC测定法在评估藻类以及可能其他样品方面的效用。
