The Microsphere Fluorescent Antioxidant (MFA) Assay: Antioxidant Activity of Spices

Increased production of reactive oxygen species is a feature of most, if not all, human disease, including cardiovascular disease and cancer. Dietary antioxidants may be especially important in protecting against human diseases associated with free radical damage to cellular DNA, lipids, and proteins. Yet, present data are not sufficient to quantify micro nutrient requirements needed to protect against oxidative damage. The antioxidant roles of many food constituents, including herbs and spices, have not been clarified. The flow cytometric Microsphere Fluorescent Antioxidant (MFA) assay measures the oxidation of R-Phycoerythrin (PE) attached to 5. 0µM microspheres via a protein coupling linker. In order to form the Radical monocation, a 25mM stock solution of ABTS (2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid free radical) was incubated 12 hr at 4C with potassium persulfate to cause removal of one electron generating a metastable ABTS cation radical (ABTS+•). This stock solution was diluted to 100µM in 50ul, which resulted in 80% quenching of 15µl of 1x10 4 PE microspheres. The antioxidant effects were measured by first incubating the 80% quenching ABTS+• with100µl portions of serial diluted antioxidants for 30 min. at RT to determine the antioxidants and concentrations that neutralized the ABTS+• Subsequently,15µl of PE-tagged microspheres were added to each sample and incubated one hour at RT. The peak fluorescent channel number was determined for 5,000 microspheres. The typical percentage protection by the antioxidant standard at 500ng/ml for Trolox (water soluble Vitamin E derivative) was 82%±0.02 SD, while Vitamin C restored 78%±0.01SD of the protective effect. For the range of 500-5,000ng/ ml, the protective effects of Trolox and Vitamin C remained constant. On the average, equivalent percentage protection for the lipid soluble fractions of 10 spice types with duplicates (courtesy of McCormick Science Institute) was not achieved until the 2,050 µg/ml levels with Cloves, Ginger, and Oregano showing the highest antioxidant protection. Interestingly, Rosemary, Sage, Thyme and Turmeric revealed a significant reduction in antioxidant activity at 2,050µg/ml. These are still significant antioxidant levels that show a secondary reason to use spices for achieving a measurable antioxidant activity. The MFA assay is very sensitive to the effects of oxidants, requires little sample, and allows application to the study of serum samples. Therefore, the antioxidant activity in healthy volunteers taking compounds to boost the antioxidant activity and to study the effects of oxidative radiation and chemotherapy may be monitored. lost during an oxidation reaction and electrons are gained in a reduction reaction. When a molecule is oxidized, another compound must gain the electron and become reduced. Therefore, redox reactions occur simultaneously to maintain a balance of electrical charge. A common biological example of oxidation and reduction occurs during the energy production process, cellular respiration. The energy produced is the result of a series of redox reactions. An example of a redox reaction is in the overall reaction of aerobic cellular respiration in utilizing glucose to produce ATP, the biological energy currency. In the overall reaction, glucose