Kevin Lowder and Dr. Tory Parker; Nutrition, Dietetics, Food Science
This project was a very meaningful experience for me, as it allowed me to work closely with my mentor with the goal of publication in a scientific journal. I had approached Dr. Parker about working in his lab prior to applying for an ORCA grant, so it was good that we already had established a rapport before working on this project.
To summarize the aims of my project with chocolate-covered strawberries, it is important to first look at the importance of antioxidants in food and then to discuss previous research in the area of antioxidant interactions and synergy.
I worked specifically with several phenolic compounds, which are naturally occurring antioxidants found in plants that can neutralize reactive oxygen species, also known as free radicals. It has been discovered that free radical action can contribute to the development of chronic disease (Battino et al., 2009). Several studies have focused on a subgroup of phenolic compounds known as flavonoids. Plumb et al. (1998) measured the individual antioxidant capacity of several flavonoids. Fernández-Pachón et al. (2004) found that the composition of flavonoids in a food sample directly correlates to the total antioxidant capacity of that food sample, though some flavonoids are more potent in their antioxidant capacity than others. Hidalgo et al. (2010) combined several flavonoids in pairs and measured antioxidant capacity. They reported synergism and antagonism between phenolic compounds, meaning that flavonoid mixtures had an antioxidant capacity that was greater than (or less than) the sum of the antioxidant capacities of individual flavonoids. Hidalgo et al. also varied the concentrations of paired flavonoids, showing that flavonoid pairing and concentration could both have an effect of overall antioxidant capacity. Previous work performed in Dr. Parker’s lab had also identified synergistic interactions between various phenolic compounds within individual fruits such as oranges, raspberries and blueberries. As the normal diet usually consists of complex mixtures various food items eaten together, the logical step was to see if mixed foods demonstrated the same synergistic interactions witnessed previously in isolated foods.
For this reason, we chose two foods that are rich in antioxidant flavonoids: dark chocolate and strawberry. Our first task was to determine if the type of chocolate used for dipping was significant, as darker chocolates contain more flavonoids. We therefore used a 54% cocoa and an 88% cocoa chocolate bar for different test groups. To determine if the ratio of chocolate to strawberry was important, we subdivided our chocolate type groups into full- and half-dips, making four total test groups. We then measured the antioxidant capacity of hydrophilic and lipophilic extracts from each experimental group, but it was found that the lipophilic extracts had almost no antioxidant activity and they were deemed unimportant for this project. After measuring antioxidant capacity of all groups and comparing them to measured values of isolated chocolate and strawberries, synergy was discovered in only the 88% fully-dipped chocolate-covered strawberry.
After discovering that synergistic interactions must be taking place at these food ratios, the next step was to pinpoint, if possible, which of the specific flavonoids within the foods were interacting. For this reason, we selected the five most abundant flavonoids between the two food items (catechin, epicatechin, pelargonidin, quercetin, p-coumaric acid) and combined them at ratios that were consistent with the 88% fully-dipped experimental group. We then measured the antioxidant capacities of the individual flavonoids and compared them against the flavonoids combinations, which consisted of every combination of two, three, four and five. These combinations were measured multiple times in triplicate at two different dilutions to ensure accuracy.
With the help of Dr. Eggett, a BYU statistician, we found statistically significant synergistic and antagonistic interactions between many of the flavonoid combinations. Interestingly, some of the interactions that were statistically significant at one dilution were insignificant at the other dilution, which reinforces the idea in addition to the total flavonoid composition of a food, both the ratio and concentration of those flavonoids plays a factor in the degree of interactions. This is an important finding that could be helpful to future researchers looking to create effective antioxidant supplements. Dr. Parker and I are currently in the process of writing a paper about the findings of this research project to submit for publication.
Overall, it was a fantastic learning experience that taught me the importance of patience and creative thinking. I encountered many setbacks during this process, including a series of assays that produced unusable data. At one point, a specially-ordered solvent was affecting the way light traveled through the solution, which drastically affected the measurements that the machine produced. In response, I was forced to try different solvents until I had found one that worked. I also made many mistakes while trying to learn the protocols for the assays that I was required to perform. While very discouraging at times to see the many hours of work and preparation produce bad data, the process was improved greatly from the help of my mentor, Dr. Parker. He helped me change my perspective and view every hour spent as a learning experience, whether or not it produced good data. Mistakes were no longer failures, but rather an inherent part of the learning process. This is a valuable perspective that I am confident will help me as I pursue further education in preparation for a career in medicine.
REFERENCES
- Battino, M.; Beekwilder, J.; Denoyes-Rothan, B.; Laimer, M.; McDougall, G. J.; Mezzetti, B. (2009), Bioactive compounds in berries relevant to human health. Nutrition Reviews, 67:S145–S150.
- Fernández-Pachón, M.S.; Villano, D; García-Parrilla, M.C.; Troncoso, A.M. (2004) Antioxidant activity of wines and relation with their polyphenolic composition. Analytica Chimica Acta 513:113–118.
- Hidalgo, M; Sanchez-Moreno, C.; de Pascual-Teresa, S. (2010), Flavonoid-flavonoid interaction and its effect on their antioxidant activity. Food Chemistry, 121:691-696.
- Plumb, G. W.; de Pascual-Teresa, S.; Santos-Buelga, C.; Cheynier, V.; Williamson, G. (1998), Antioxidant properties of catechins and proanthocyanidins: Effect of polymerisation, galloylation and glycosylation. Free Radical Research, 29:351–358.