Antioxidant Activity in Various Oxidative Environments
In this section, the antiradical capacities of alkyl gallates and gallic acid were examined using different chemical methods: β-carotene bleaching, DPPH scavenging, and Rancimat assays. These assays were carried to evaluate the impact of -COOH group and the ester chain length on the antioxidative activity of gallic acid derivatives in various oxidative environments.
The ability of antioxidative compounds for scavenging DPPH free radicals is presented in Table 2. The antiradical activities of gallic acid and alkyl gallates enhanced as the antioxidant concentrations increased from 10 to 50 μM. The percent values of for gallic acid at all concentrations were significantly higher than the alkyl gallates. Considering the IC50, the gallic acid concentration required to scavenge 50% of DPPH was considerably lower than its alkyl ester derivatives. These results were in accordance with Asnaashari et al. (2019) and Mansouri et al. (2020) that reported the anti-DPPH• activity of gallic acid was significantly more potent compared to methyl gallates.
The O-H bond disassociation enthalpy (O-H BDE), intramolecular hydrogen bonds (IHB), and ionization potential are the three critical parameters that affect the antiradical activity of antioxidant agents. Given that the O-H BDE, IHB, and ionization potential of alkyl gallates are similar, thus the steric impact can be a plausible explanation for the difference in antiradical activity of alkyl gallates. The ester structure affects the polarity and solubility of antioxidant compounds as well as their molecular mobility and availability to oxidative components (Lu et al., 2006). Our results showed that the radical scavenging potency of gallic acid as affected by an extra carboxyl group than alkyl gallates. In fact, the dissociation possibility of the carboxyl group in polar media is the main reason for increased the electron-donating and H-atom transfer activities of gallic acid than the alkyl gallates. In contrast to the electron-donating impact of carboxylate anion, -COOH is considered an electron-withdrawing group that increases the O-H BDE of the phenolic ring (Correia et al., 2004). As can be seen in Table 2, the polarity (log P) of gallic acid was significantly higher than the alkyl gallates, which is leading to more hydrophilic interactions between the antioxidant and the polar molecules of methanol as a reaction solvent.
In the β-carotene bleaching assay, gallic acid and alkyl gallates prevented the extent of β-carotene bleaching by neutralizing the linoleate-free radical and other free radicals formed in the emulsion. As shown in Table 2, a concentration-dependent antioxidant potential was observed for all antioxidant components studied. A comparison of antioxidant capacity in the emulsion system showed that gallic acid was the lowest reactive antioxidant than alkyl gallates. So that, the IC50 value of antioxidant agents was as follows: dodecyl gallate < octyl gallate < propyl gallate ~ methyl gallate < gallic acid < stearyl gallate. This means that the alkyl gallates concentration required for scavenging %50 of linoleate-free radicals was significantly lower than the gallic acid. In general, due to the appropriate length of the alkyl chain, dodecyl gallate had a more effective interfacial performance at the water-oil interfaces. The nonlinear behavior or cut-off effect was observed for inhibitory activities of homologous series of antioxidative compounds in the W/O emulsion. The interfacial activities of gallic acid derivatives enhanced as the lengths of the alkyl chain were reached until a critical chain length (12 carbon atoms); after that, further chain length extension causes a decrease in antioxidant activities. Medina et al. (2009), Laguerre et al. (2010) and Meireles et al. (2019) reported similar findings showing that the lipophilic esters with short medium-chain compared to long-chain esters improved the activity of antioxidative compounds in the multiphase system. Laguerre et al. (2009) also reported in the emulsified medium the antiradical potency enhances as the alkyl chain is lengthened, with a threshold for the dodecyl chain, after which further chain extension causes a drastic decrease in antiradical activity. The increased antiradical activities of the alkyl gallates in β-carotene-linoleic acid emulsion compared to gallic acid can be attributed to increased hydrophobicity (log P), surface-active characteristic, and the precise placement of antioxidants in the water-oil interface.
In the Rancimat test, the oxidative stability index (OSI) of stripped soybean oil (0.32 h) was significantly promoted with the antioxidants added. The OSI values of bulk soybean oil treatments with antioxidant agents were as follow: propyl gallate > octyl gallate ~ dodecyl gallate > stearyl gallate ~ methyl gallate > gallic acid. A higher induction period was obtained for soybean oil containing propyl gallate compared to other antioxidants means that propyl gallate was the most effective antioxidant to increase oil oxidative stability at high temperature (120 °C). This result was in accordance with Phonsatta et al. (2017) that reported the OSI of bulk oil containing alkyl gallates, especially propyl gallate, was significantly more potent compared to gallic acid. Concerning the carboxyl group’s electron-withdrawing character, which is not markedly dissociated in the hydrophobic media, gallic acid had the lowest inhibitory activity than alkyl gallates in the lipid system. Studies have shown that the esterification of gallic acid with fatty alcohols can be increasing its hydrophobicity as well as surface-active properties (Chaiyasit et al., 2005). According to log P results (Table 2) the hydrophobicity of alkyl gallates was higher than the gallic acid. Therefore, the increased antioxidant activities of the alkyl gallates, especially propyl gallate, in bulk phase oil compared to gallic acid can be attributed to improved amphiphilic properties by increasing the alkyl chain length and the precise placement of antioxidants in the actual site of oxidation. Our findings agreed with those reported by Kikuzaki et al. (2002). The results clearly showed the nonlinear antioxidative activities for alkyl gallates due to enhance in the length of the alkyl chain. The size of antioxidants affects their activity by changing their mobility in the bulk phase oils, leading to a cut-off effect. Lipophilic antioxidative components by long alkyl chains have lower mobility than those with a smaller size because of steric hindrance, so decreased diffusibility toward the reactive centers such as free radicals and oxidizable substrates. Furthermore, enhance in the length of the alkyl chain increase the possibility of hydrophobic interaction among antioxidative components and the environment such as the membrane or emulsifiers, which cause a decrease in mobility and diffusion of the antioxidative components to the oxidation site of bulk oil (Laguerre et al., 2015).