Eggplant Phenolamides: 2-Nonenal Scavenging and Skin Protection Against Aging Odor

Abstract

Eggplants are high in polyphenols, making them a powerful antioxidant food that is beneficial for health and has excellent anti-aging effects. As metabolism slows down with aging, lipid peroxides are generated, with 2-nonenal being the main cause of old-age odor, which has a detrimental effect on skin keratinocytes. In this study, the 2-nonenal scavenging ability of fruits, leaves, stems, and roots of eggplant was evaluated, and the active compound was identified as N-trans-feruloylputrescine. Furthermore, we assessed whether the extracts and N-trans-feruloylputrescine showed a protective effect against skin damage induced by 2-nonenal. The antioxidant activity of the eggplant extracts was evaluated using DPPH and ABTS assays, and the fruits exhibited stronger antioxidant activity compared to the other extracts. Additionally, it was found that the ROS levels increased by 2-nonenal were significantly reduced by eggplant fruits and roots, which also inhibited lipid peroxidation. These results suggest the possibility of inhibiting the production of 2-nonenal itself. These findings suggest that eggplant extracts and the N-trans-feruloylputrescine can have a positive effect on preventing aging and maintaining skin health.

Keywords: 2-nonenal; N-trans-feruloylputrescine; aging-odor; anti-aging; eggplant; keratinocytes; phenolamide.

Figure 1

Antioxidant activity of eggplant extract evaluated by DPPH and ABTS assays. The results are expressed as ascorbic acid equivalents per gram of dry weight (μg ascorbic acid/g extract). These data are expressed as mean ± SD of three independent experiments.

Figure 2

Effects of eggplant extracts on arachidonic acid reduction induced by iron/ascorbic acid-mediated lipid peroxidation. Arachidonic acid (2 mM) was subjected to lipid peroxidation induced by 250 µM FeSO₄ and 2.5 mM ascorbic acid as oxidative triggers, with or without the addition of 2 mg/mL eggplant extracts. The control group consisted of arachidonic acid alone, without oxidative inducers. The reaction mixture was incubated at 37 °C for 1 h and then analyzed by HPLC to quantify the residual arachidonic acid. Data are expressed as the mean ± SD of 3 independent experiments. ^### p < 0.001 vs. control; *** p < 0.001 vs. 2-nonenal.

Figure 3

2-nonenal scavenging activity of eggplant extracts from different parts and compositional changes in fruits after reaction with 2-nonenal. (A) 2-nonenal (0.1 mM, in ethanol) was incubated with eggplant extracts from the fruits, leaves, stems, and roots at different concentrations (1, 5, and 10 mg/mL) in PBS at 37 °C for 24 h. After incubation, the reaction mixtures were analyzed by HPLC to quantify the residual amount of 2-nonenal. Data are presented as the mean ± SD of 3 independent experiments. *** p < 0.0001 vs. control. (B) 2-nonenal (10 mM, in ethanol) was incubated with 10 mg/mL fruits at 37 °C for 72 h. As a control, the extract was incubated with an equivalent volume of ethanol. After incubation, chromatograms were comparatively analyzed between the fruit extract alone and the reaction mixture with 2-nonenal. (C) The fruits (10 mg/mL) were incubated with various concentrations of 2-nonenal (1, 5, and 10 mM) at 37 °C for 24 h, and HPLC analysis was performed to assess differences in compound reduction depending on 2-nonenal concentration. Peaks marked with an asterisk (*) indicate compounds that decreased after the reaction. (D) Chemical structures corresponding to the peaks labeled in Figure 3B,C.

Figure 4

Analysis of 2-nonenal scavenging activity and proposed reaction mechanism of N-trans-feruloylputrescine (3). (A) 2-nonenal (0.1 mM, in ethanol) was incubated with N-trans-feruloylputrescine (3) at different concentrations (1, 5, and 10 mM) in PBS at 37 °C for 24 h. After incubation, the reaction mixtures were analyzed by HPLC to measure the residual amount of 2-nonenal. Data are presented as the mean ± SD of 3 independent experiments. *** p < 0.001 vs. control. (B) 2-nonenal (10 mM) was incubated with or without 10 mM N-trans-feruloylputrescine (3) at 37 °C for 72 h. As a control, N-trans-feruloylputrescine (3) was incubated with an equivalent volume of ethanol. After incubation, chromatographic profiles were compared between compound 3 alone and its reaction mixture with 2-nonenal. Peaks marked with an asterisk (*) indicate newly formed adducts resulting from the reaction. (C) Proposed mechanism of covalent adduct formation between 2-nonenal and N-trans-feruloylputrescine (3).

Figure 5

Effects of eggplant extracts and N-trans-feruloylputrescine (3) on 2-nonenal-induced damage in human keratinocytes. (A) Cell viability of HaCaT cells pre-treated with eggplant extracts (fruits, leaves, stems, and roots) at various concentrations (1, 5, and 10 µg/mL) for 2 h, followed by exposure to 2-nonenal (100 µM) for 24 h. (B) Cell viability of HaCaT cells pre-treated with N-trans-feruloylputrescine (3) at concentrations ranging from 5 to 200 µM for 2 h, followed by exposure to 2-nonenal (100 µM) for 24 h. These data are expressed as mean ± SD of 3 independent experiments. ^### p < 0.001 vs. control; * p < 0.05, ** p < 0.01 *** p < 0.001 vs. 2-nonenal.

Figure 6

Effects of eggplant extracts on ROS production in 2-nonenal-induced damage in human keratinocytes. (A) Cellular ROS production was analyzed using the CM-H2DCFDA dye and observed under a fluorescence microscope (scale bar = 250 µm). HaCaT cells were pre-treated with eggplant extracts (10 µg/mL) for 2 h, followed by exposure to 100 µM 2-nonenal for 24 h. (B) Quantification of ROS levels was performed using ImageJ software (version 1.48) by measuring CM-H2DCFDA-positive fluorescence intensity in at least 5 randomly selected fields per well across 3 independent experiments. Data are expressed as mean ± SD from 3 independent experiments. ^### p < 0.001 vs. control; ** p < 0.01, *** p < 0.001 vs. 2-nonenal.