Joshua Reber and Dr. Tory Parker, Dietetics and Food Science
Epidemiological evidence has shown, on average, that those who consume more servings of fruits and vegetables have lower relative risk for cardiovascular disease and some cancers (Lee et al., 1991; Hertog et al., 1993; van’t Veer et al., 2000). As a result, a great deal of research has explored the components of fruit (Robards et al., 1999), with primary emphasis being placed on compounds in the phenolic category. This focus developed from the high in vitro antioxidant capacity of these compounds and the potential for them to protect lipids, protein and DNA from oxidative damage (van’t Veer et al., 2000). The high antioxidant capacity of fruit and phenolics in vitro suggests that antioxidant capacity might explain the epidemiological evidence.
There is a large discrepancy between the antioxidant capacity of an individual antioxidant at the concentration found in fruit and the antioxidant capacity of the whole fruit (compare Wu et al., 2004 and Tabart et al., 2009). The antioxidant capacity of the whole fruit is much lower. The intent of this study was to quantify the endointeractions of a number of the individual antioxidants at the concentrations found in strawberries. Our hypothesis was that phenolic compounds would function most effectively in an antioxidant complex with other phenolic compounds. However, antagonism was also hypothesized as a possible explanation for the discrepancy between the antioxidant capacities of individual phenolics vs. the whole fruit. Testing the antagonistic, additive or synergistic antioxidant capacity of phenolic compounds at the concentrations present in strawberries would give us a better understanding of complex interactions that might contribute to the nutritional value of strawberries.
Using a protocol previously described by Dr. Parker, I began to perform the Oxygen Radical Absorbance Capacity (ORAC) assay on the individual phenolic compounds found in strawberries. The ORAC assay is widely used and accepted as a valid antioxidant capacity assay. To briefly explain, the samples are diluted to fit a Trolox (water-soluble vitamin E analog) standard curve. The samples are injected into a 96 well plate containing a radical generator (AAPH) and fluorescein. A fluorometer detects the fluorescence of the fluorescein over a two hour time period. As the fluorescein is oxidized by the AAPH, it slowly decreases its ability to fluoresce. The sample of antioxidant rich fruit extracts protects the fluorescein, allowing it to fluoresce for a longer period of time. The area under the curve is measured and used to give an ORAC value for the sample which can then be used for statistical analysis. Once the antioxidant capacity of each individual phenolic compound had been determined, we were able to move on to each of the possible combinations of two for all of the individual compounds.
The ORAC values found for each combination of two was compared to the sum of the individual phenolics to determine whether the combination was additive, synergistic or antagonistic. Those combinations of two that were found to be synergistic were carried on to the next phase of testing, where all possible combinations of three that included the previously concluded synergistic combination of two were assayed. Again, the value for the combination of three was compared to the sum of the three individual compounds.
A similar pattern was followed for combinations of four. There were no combinations of four that were found to be synergistic.
Overall, it was found that the different combinations varied greatly in whether they produced an additive, synergistic or antagonistic effect. There were many combinations that exhibited great synergism, while others exhibited great antagonism. Certain patterns were noticed within these results, such as repeatedly finding many of the same compounds contained in either the synergistic combinations or the antagonistic combinations, something that was very exciting to see.
Though the project gave us some insight as to what is going on in the fruits we eat, it also opened my eyes to the complexities of the interactions that occur within them. Many factors affect antioxidant status, and much, much more research is needed before we completely understand what is going on in our food. We are currently moving into a new phase of research involving supplementation of phenolic compounds in humans, and I am also involved in a study searching for an anti-cancer antioxidant relationship.
The mentoring experience overall has been very positive. I feel that I have learned more doing research about the scientific method than I have in all of my science classes combined. The principles and methods of research I have learned from Dr. Parker will be carried with me and will always be looked back upon as a very influential part of my life and education.
I will be forever grateful to the donors who make the mentoring experience possible. Their generosity has allowed me to explore parts of life and education that I had never before considered.
References
- Robards, K.; Prenzler, P. D.; Tucker, G.; Swatsitang, P.; Glover, W. Phenolic compounds and their role in oxidative processes in fruits. Food Chem. 1999, 66, 401-436.
- Lee, H. P.; Gourley, L.; Duffy, S. W.; Esteve, J.; Lee, J.; Day, N. E. Dietary effects on breast-cancer risk in Singapore. Lancet 1991, 337, 1197-1200.
- Hertog, M. G.; Feskens, E. J.; Hollman, P. C.; Katan, M B.; Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 1993, 342, 1007-1011.
- van’t Veer, P.; Jansen, M. C. J. F.; Klerk, M.; Kok, F. J. Fruits and vegetables in the prevention of cancer and cardiovascular disease. Public Health Nutr. 2000, 3, 103-107.
- Tabart J.; Kevers C.; Pincemail J.; Defraigne J.-O.; Dommes J. Comparative antioxidant capacities of phenolic compounds measured by various tests. Food Chem. 2009, 113, 1226-1233
- Wu, X.; Gu, L.; Holden, J. M.; Haytowitz, D. B.; Gebhardt, S. E.; Beecher, G. R.; Prior, R. L. Development of a database of total antioxidant capacity in foods: a preliminary study. J. Food Comp. Anal. 2004, 17, 407-422.