Unexpected Antioxidant Efficiency of Chlorogenic Acid Phenolipids in Fish Oil-in-Water Nanoemulsions: An Example of How Relatively Low Interfacial Concentrations Can Make Antioxidants to Be Inefficient

Abstract

Selecting effective antioxidants is challenging since their efficiency in inhibiting lipid oxidation depends on the rate constants of the chemical reactions involved and their concentration at the reaction site, i.e., at the interfacial region. Accumulation of antioxidants at the interface of emulsions is key to modulate their efficiency in inhibiting lipid oxidation but its control was not well understood, especially in emulsions. It can be optimized by modifying the physicochemical properties of antioxidants or the environmental conditions. In this work, we analyze the effects of surfactant concentration, droplet size, and oil to water ratio on the effective interfacial concentration of a set of chlorogenic acid (CGA) esters in fish oil-in-water (O/W) emulsions and nanoemulsions and on their antioxidant efficiency. A well-established pseudophase kinetic model is used to determine in the intact emulsified systems the effective concentrations of the antioxidants (AOs). The relative oxidative stability of the emulsions is assessed by monitoring the formation of primary oxidation products with time. Results show that the concentration of all AOs at the interfacial region is much higher (20-90 fold) than the stoichiometric one but is much lower than those of other phenolipid series such as caffeic or hydroxytyrosol derivatives. The main parameter controlling the interfacial concentration of antioxidants is the surfactant volume fraction, Φ_I, followed by the O/W ratio. Changes in the droplet sizes (emulsions and nanoemulsions) have no influence on the interfacial concentrations. Despite the high radical scavenging capacity of CGA derivatives and their being concentrated at the interfacial region, the investigated AOs do not show a significant effect in inhibiting lipid oxidation in contrast with what is observed using other series of homologous antioxidants with similar reactivity. Results are tentatively interpreted in terms of the relatively low interfacial concentrations of the antioxidants, which may not be high enough to make the rate of the inhibition reaction faster than the rate of radical propagation.

Keywords: antioxidants; chlorogenic acid; distribution; droplet size; emulsion; interfacial concentration; lipid oxidation; nanoemulsion; phenolipids; pseudophase kinetic model.

Figure 1

Representation of aqueous, oil, and interfacial regions of an emulsion showing approximate locations of surfactant and of reactive group of hydrophobic ArN₂⁺ ions, as well as distribution of an antioxidant. P_O^I, partition constant of AO between oil-interfacial region; P_W^I partition constant of AO between aqueous-interfacial region, k_I, rate constant for reaction between 16-ArN₂⁺ and AO in interfacial region. Subscripts O, I, and W indicate oil, interfacial, and aqueous regions, respectively, and Φ is volume fraction of a region (Φ = V_region/V_total).

Figure 2

Percentages (%AO) of CGA and its esters in different regions (O—oil; I—interface; W—water) of 1:9 (O/W) nanoemulsions (A–C) and 4:6 (O/A) emulsions (D–F) (fish oil/0.04 M citrate buffer, pH = 3.65/Tween 80), T = 25 °C.

Figure 3

Effective concentration of chlorogenic acid (CGA) and its esters in interfacial (I), oil (O) and aqueous (W) regions of 1:9 (O/W) nanoemulsions (A–C); [AO_T] = 0.125 mM) and 4:6 (O/W) emulsions (D–F); [AO_T] = 0.500 mM).

Figure 4

Interfacial concentration (AO_I) for different phenolipids series in 1:9 (O/W) nanoemulsions ([AO_T] = 0.125 mM) AG, gallic acid serie; HT, hydroxytyrosol serie. Data for AG and HT series obtained from references [21,28].

Figure 5

(A) Interfacial concentrations of CGA and CGA esters in 1:9 (O/W) emulsions (●—Φ_I = 0.005, ■—Φ_I = 0.01, ▲—Φ_I = 0.02) and 1:9 (O/W) nanoemulsions (—Φ_I = 0.005, —Φ_I = 0.01, —Φ_I = 0.02) at different emulsifier volume fractions. (B) Effect of O/W ratio on interfacial AOs concentration in nanoemulsions at Φ_I = 0.005. In both graphs, [AO_T] = 0.125 mM.

Figure 6

Effects of AO chain length at Φ_I = 0.005, 0.01, and 0.02 on oxidative stability of fish oil 4:6 O/W emulsions (A) and 1:9 (O/W) nanoemulsions (B) in terms of time needed to reach an increase of 0.5% in diene conjugated content. Comparison between antioxidant efficiency of AOs in 4:6 O/A emulsions (C) and 1:9 O/A nanoemulsions (D) and values of (AO_I) in same systems at Φ_I = 0.005, 0.01 and 0.02.

Scheme 1

Representation of rate-limiting step of initiation and propagation steps of lipid oxidation reaction (1)–(3) and of competing inhibition reaction in presence of antioxidants (4). LH: Lipid; LOO^•: peroxyl radical; L^•: lipid radical; LOOH: lipid hydroperoxide; ArO-H: antioxidant and ArO^•: antioxidant radicals.

Scheme 2

Representation of the oxidation reactions involving antioxidants (ArOH). LH: Lipid; LOO^•: peroxyl radical; L^•: lipid radical; LOOH: lipid hydroperoxide; ArO-H: antioxidant and ArO^•: antioxidant radicals.