Richard Low and Dr. Tory Parker, Department of Nutrition, Dietetics, and Food Science

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According to a review by Battino et al. (2009), blueberry consumption can regulate blood pressure, inhibit cancer growth, and improve cardiovascular health by preventing plaque buildup in the arteries. Naturally occurring antioxidants found in fruits and vegetables that are thought to stop the chain reactions of harmful free radicals that could lead to chronic disease (Battino et al., 2009). However, human trials testing individual antioxidants supplements found in fruit have not conclusively demonstrated chronic disease prevention (Herrera et al. 2009). Dr. Parker and I hypothesized that one reason whole fruit provides more antioxidant protection than individual antioxidant supplements is that antioxidants work together synergistically.

To test this hypothesis, I first found the reported concentrations of different antioxidants in blueberries. We then purchased seven of these compounds and tested their individual antioxidant strength using the Oxygen Radical Absorbance Capacity (ORAC) assay. This assay measures the ability of the antioxidant to protect a target molecule from free radicals. Each run of the assay takes around 3 hours to complete for each 96 well plate. This assay was difficult for me to master and many of my early plates had to be redone because of mistakes. Eventually I obtained data for each of the seven blueberry antioxidants.
After determining the antioxidant strength of the individual compounds, I combined the single compounds into groups of two to see which combinations were synergistic. There were 21 different combinations of two and after doing the combinations of two we were planning on doing the combinations of three and four compounds, which would have been 70 more combinations. However the results from the sets of two did not give us significant results.

Upon analyzing the data, we realized that we had two problems with our combinations of two. First, we realized that the ORAC assay was giving inconsistent results on regions of our 96 well plate. To solve this we stopped using the regions of the plate that were ineffective and determined to run extra plates to give us more data points. Another realization was that perhaps the ratios of the antioxidants to each other affected their synergy. In our first set of combinations of two, we did not pay attention to the ratios at which we combined the antioxidants, we simply combined them at concentrations that would fit our standard curve. To address this issue of ratios, we determined to run the data again with the ratios of antioxidants found in blueberries and a 1:1 control ratio.

Unfortunately some of the antioxidants we used were very expensive, costing up to $100 per mg. So to continue with our study we had to eliminate two compounds from our set of seven antioxidant compounds. With the remaining five compounds, we found the ORAC values of the combinations at fruit ratios as well as at a 1:1 ratio. This time we also ran three times as many plates in order to overcome the variability of the ORAC assay and establish statistical significance.

This time we found significant synergy between almost all of our antioxidants. With some of the combinations the fruit ratio was more synergistic than the 1:1 ratio, while with others the 1:1 ratio was better than the fruit ratio. We are now more interested in the specific ratios that produce optimal synergy and I will continue this line of research this year.

I presented my findings at the July, 2010 International Food Technologists (IFT) conference in Chicago, IL. For me this was a very fulfilling and helpful opportunity, as I was exposed to other research in the field of nutrition and was able to connect with others doing similar research to mine.
Dr. Parker and I are now preparing to submit our results to a peer reviewed, scientific journal. We hope to have these results published by the end of the Winter 2011 semester. We are also in the process of patenting our combinations through the BYU patents office. In addition, two other students in Dr. Parker’s lab are going to be conducting a human blueberry study this upcoming year using some of the blueberry data from my project.

Overall, conducting original research has been both challenging and rewarding. I am grateful to have contributed to the understanding of nutrition and the human body.

Sources

• Battino, M.; Beekwilder, J.; Denoyes-Rothan, B.; Laimer, M.; McDougall, G.J.; Mezzetti, B. Bioactive compounds in berries relevant to human health. Nutr. Rev. 2009, 67, S145-S150.
• Herrera, E.; Jimenez, R.; Aruoma, O.I.; Hercberg, S.; Sanchez-Garcia, I.; Fraga, C. Aspects of antioxidant foods and supplements in health and disease. Nutr. Rev. 2009, 67, S140-S144.