Investigation of Radical and Dicarbonyl Scavenging Potential of Raw and Processed Foods During Simulated Gastrointestinal Digestion

Abstract

Oxidative and dicarbonyl stresses generated by the imbalance between the metabolism or the generation of the free radicals and α-dicarbonyl compounds are associated with chronic diseases. Biologically active pure compounds found naturally in foods come into prominence with their ability to prevent diseases by counteracting against free radicals and α-dicarbonyl compounds; however, foods, the multi component systems that contain varying amounts of these compounds, should be also investigated in detail. In addition to being exposed to the mentioned stresses mainly through the dietary derived free radicals and α-dicarbonyl compounds, the gastrointestinal tract is of great importance as being one of the major carriers of dietary scavenging compounds in the human body. Although a substantial body of research is interested in the potential roles of foods on oxidative stress in the human body, dicarbonyl stress and its inhibition by foods is a new topic to examine in detail. In this framework, the main objective of this PhD thesis was to investigate the radical and dicarbonyl scavenging potential of foods under simulated gastrointestinal conditions. To that end, most consumed raw and processed foods in the daily diet were evaluated in terms of their antioxidant and methylglyoxal scavenging activity. The antioxidant capacity of most consumed foods in the daily diet was investigated under simulated gastrointestinal conditions and the possible interactions (synergism, antagonism, and additive) between co-consumed foods were evaluated in the first part of this dissertation. Total antioxidant capacities of the selected foods were monitored at different stages of in vitro simulated gastrointestinal digestion system and in their bioaccessible fractions. According to the statistical comparison between estimated and observed data, the resultant interaction types were determined at each step. Slightly alkaline conditions were found to significantly (p<0.05) increase the total antioxidant capacity of foods. Synergism was observed during the digestion of the combinations of milk with fruits or tea extracts, which were thought to be most likely due to the stabilization and regeneration of phenolic compounds by proteins. Hydroxyl radical scavenging capacity was also determined in the bioaccessible fractions of food to show the antioxidant capacity of foods with a physiological resembling approach. Green tea extract was found to be the most efficient scavenger (936.48 ± 16.64 mmol TEkg-1) to eliminate hydroxyl radicals followed by black tea extract with 508.49 ± 12.15 mmol TEkg-1 of hyrodxyl radical scavenging capacity. As there is a lack of a standardized method for measuring the dicarbonyl scavenging activity of compounds, a new approach to determine the methylglyoxal scavenging activity was described in this thesis. After the kinetic evaluation of the reaction between methylglyoxal and the possible scavenging compounds, amino, thiol, and phenolic compounds, under physiological conditions (37 °C, pH 7.4), methylglyoxal scavenging activity of the examined scavenging compound was calculated by the area under the curve of the plot showing the methylglyoxal inhibition percentage as a function of time. Cysteine equivalent methylglyoxal activity (CEMSA) was obtained by dividing the resultant area under the curve for the scavenging compound by that of cysteine to express the methylglyoxal activity as an equivalent activity of the reference compound. Accordingly, the CEMSA values of some selected compounds were in the following order; phloretin > epicatechin > cysteine > epigallocatechin gallate > gallic acid > creatine > hesperetin > resveratrol > phloridzin > quercetin > chlorogenic acid > naringin > genistein > tryptophan. To apply this methodology to foods, the bioaccessible fractions of certain food groups including nuts, seeds, legumes, meat, dairy products, fruits, brassica, and allium vegetables, obtained after gastrointestinal digestion were reacted with methylglyoxal under physiological conditions over 24 h. Among foods, nuts, meat, egg, cheese, strawberry, broccoli, garlic, and onion were found to have CEMSA values higher than 15 mmol cysteine equivalent per kg. The CEMSA values were positively correlated (r > 0.526) with total thiol and amino groups for all foods except fruits. Moreover, the standard compounds, beverages, and foods were categorized according to their methylglyoxal inhibition percentages and rates as "rapidly reacting with high capacity", "slowly reacting with high capacity", and "slowly reacting with low capacity". Finally, the percentages that meet the daily required methylglyoxal scavenger need of each food were calculated. Considering the methylglyoxal elimination by a multi component system including scavenging compounds being together in the human plasma, the interactions between dicarbonyl scavenging compounds were investigated under physiological conditions. Methylglyoxal inhibition in the presence of the binary combinations of the most efficient dicarbonyl scavenging compounds (epicatechin or cysteine) with certain other dicarbonyl scavengers was kinetically investigated under physiological conditions. Using an irreversible bimolecular reaction model, the estimated and observed reaction rate constants in the binary combinations of scavenging compounds were calculated. According to the comparison between the observed and estimated reaction rate constants, the interaction types (synergism, antagonism, and additive) between certain scavenging compounds were determined. As the observed rate constant (ko=1.81±0.17 Lmmol-1h-1) for the reaction between cysteine and epicatechin was found to be significantly greater (p<0.05) than the estimated rate constant (ke=1.08±0.11 Lmmol-1h-1), synergism was observed in this combination. To evaluate the potential side reactions, dicarbonyl scavengers were also incubated without methylglyoxal. Epicatechin was found to antagonistically interact with scavengers such as creatine, quercetin, and gallic acid that stimulate its oxidation during methylglyoxal scavenging. In addition to the methylglyoxal scavenging activity of different foods widely consumed in the daily diet determined under physiological conditions in the second part of this thesis, their methylglyoxal scavenging potential was also investigated under simulated gastrointestinal conditions. Four food groups having different arginine, creatine, thiol, and flavonoid contents were subjected to simulated gastric and intestinal conditions together with methylglyoxal. In comparison to control, foods from group 1 (chicken, beef and egg), group 2 (walnut, hazelnut, and kidney bean) and group 3 (broccoli, onion, garlic, and cauliflower) caused a significant (p<0.05) decrease in the concentration of methylglyoxal under gastric conditions. Egg was found as the most efficient methylglyoxal scavenger food under gastric conditions. All foods caused significant (p<0.05) decreases in the concentration of methylglyoxal under intestinal conditions. Chicken, beef, and broccoli were found to scavenge more than 80% of methylglyoxal during 2h of intestinal digestion. Changes in the concentration of methylglyoxal were monitored kinetically during the intestinal phase. The reaction of methylglyoxal with scavenging compounds in foods was evaluated using an irreversible bimolecular reaction model. Reaction rate constants and initial reaction rates were calculated for each food. The highest reaction rate constant was estimated as 26.6±1.38 Lmol-1min-1 for egg, while the highest initial reaction rate was 3.6±0.42 mMmin-1 for chicken. Foods were ranked according to their methylglyoxal scavenging rates under intestinal conditions and their scavenging potential was associated with their scavenging content. The effects of different cooking methods on the methylglyoxal scavenging potential of meat, that was found to be one of the most efficient foods to scavenge methylglyoxal according to the previous results obtained from the previous section, were investigated under simulated gastrointestinal conditions. Grilling and sous vide cooking were chosen as representative cooking techniques for high temperature short time and low temperature long time heating, respectively. Beef and chicken meat grilled and sous vide cooked at different degrees of doneness were subjected to simulated gastric and intestinal digestion with methylglyoxal. Comparing to raw meat, grilling, and mincing were found to reduce methylglyoxal scavenging potential of beef samples, by 7% and 23% respectively. However, no difference (p>0.05) was observed in methylglyoxal scavenging potential of beef and chicken meats after sous vide cooking. Among the compositional factors (thiol groups, free amino groups, creatine, arginine, and lysine), creatine in raw and grilled beef was found to be the most significant methylglyoxal scavenger under simulated intestinal conditions.