The antioxidant and anti-adipogenic properties of phenolic acids identified in Mageu

Abstract

The increasing burden of non-communicable diseases (NCDs), such as obesity and cardiovascular diseases, poses significant public health challenges across African countries. Identifying functional foods with health-promoting properties has become a priority in addressing these issues. Mageu, a fermented maize-based beverage native to Southern Africa, is of particular interest due to its widespread consumption and cultural significance. Beyond its traditional role in the diet, Mageu has been shown to possess bioactive properties, including cellular antioxidant, anti-adipogenic, and anti-inflammatory effects, largely attributed to its phenolic compounds namely, ferulic acid (FA), caffeic acid (CA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 4-hydroxybenzoic acid (4-HBA), and p-coumaric acid (p-CA).. Simulated in vitro digestion studies have revealed increased concentrations of FA, 3,4-DHBA and 4-HBA. Given the prevalence of NCDs in Southern Africa, this study aimed to evaluate the antioxidant, anti-adipogenic, cytotoxicity and the related polyphenol-polyphenol interactions of 3,4-DHBA, 4-HBA, and FA, the most abundant phenolic acids identified in Mageu. This research is crucial for understanding the potential health benefits of Mageu and its role in mitigating the growing health crisis in the region. The effective concentration at which 3,4-DHBA, 4-HBA, and FA neutralized reactive oxygen species (ROS) by 50% (half maximal effective concentration (EC50)) was determined with the oxygen radical absorbent capacity (ORAC) assay. Using this information the antioxidant properties at the EC25 and EC50, (EC50(2X) and EC50(5X) for some assays) were determined for each phenolic acid and combinations thereof with the Folin-Ciocalteu (F-C) assay, Trolox equivalent antioxidant capacity (TEAC) and ORAC assays. Then inhibition of oxidative damage was demonstrated using the dichlorofluorescein diacetate (DCFH-DA) cellular (Caco-2 cells) antioxidant assay. Dosage dependent differences in activity was observed in most instances. Although not significant, FA consistently had the highest antioxidant activity, when compared with 3,4-DHBA and 4-HBA. Structural differences, particularly the arrangement and number of hydroxyl groups on the phenolic ring, contributed to these differences in antioxidant capacity. Synergism was observed at the EC25 with the F-C assay for 3,4-DHBA/4-HBA and 3,4-DHBA/FA for the TEAC assay, and with the ORAC assay for 3,4-DHBA/4-HBA/FA. With the DCFH-DA assay synergism was observed at the EC50 for 3,4-DHBA/4-HBA, 4-HBA/FA and 3,4-DHBA/4-HBA/FA, at EC50(X2) for 3,4-DHBA/4-HBA/FA and at EC50(X5) for 3,4-DHBA/4-HBA and 3,4-DHBA/4-HBA/FA. The efficacy of these polyphenols and their combinations in mitigating lipid accumulation in 3T3-L1 differentiated to adipocytes cells assessed using Oil Red O (ORO) and Nile Red (NR) assays. Phenolic acids demonstrated significant dose-dependent anti-adipogenic effects at elevated concentrations relative to control cells in both assays. At EC25, EC50, and EC50(X2) concentrations, polyphenol-polyphenol interactions were predominantly additive. Notably, synergistic interactions were consistently observed in triple combinations and at higher concentrations. Synergy was particularly evident in the ORO assay at EC25 and EC50(X5) for the combination of 3,4-DHBA/4-HBA/FA. In the NR assay, synergy was observed at EC25 for 3,4-DHBA/4-HBA/FA, at EC50(X2) for 3,4-DHBA/FA, 4-HBA/FA, and 3,4-DHBA/4-HBA/FA, and at EC50(X5) for 3,4-DHBA/FA and 3,4-DHBA/4-HBA/FA. These phenolic acids also synergistically inhibited the differentiation of preadipocytes, thereby preserving their fibroblast morphology. Finally, the cytotoxicity was determined in the Caco-2 and 3T3-L1 cell lines with the Crystal Violet (CV) assay. At the EC25 in the Caco-2 cell, the combination of 4-HBA and FA significantly reduced the percentage cell number to 83.7 ± 1.88% after 24 hours, but not at 48 and 72 hours. Similarly, 3,4-DHBA with 4-HBA and FA reduced the % cell number to 81.23 ± 4.69% after 24 hours, with no significant effects at 48 and 72 hours. At the EC50, 3,4-DHBA, 4-HBA, and FA were not cytotoxic, but combinations of 3,4-DHBA + 4-HBA, 3,4-DHBA + FA, and 4-HBA + FA decreased the percentage cell biomass after 24 hours, with lesser effects after 48 and 72 hours. Differences between the EC25 and EC50 groups were not statistically significant. The cytotoxic effects of phenolic acids on preadipocyte mouse fibroblasts, 3T3-L1 cells were time- rather than dose-dependent, with mild cytotoxicity observed at 72 hours. The observed anti-adipogenic effects were not directly linked to cytotoxic activity. Notably, mixtures containing 4-HBA, particularly at lower concentrations, inhibited the proliferation of the Caco-2 and 3T3-L1 cells. In conclusion, Mageu derived phenolic acids possess both antioxidant and anti-adipogenic properties, with their effects modulated by concentration and influenced by additive and synergistic interactions. These elucidate the potential health benefits of Mageu and its phenolic acid constituents, contributing to the understanding of Mageu as a source of bioactive compounds with therapeutic potential.