Antioxidant Activity of Compounds Isolated from Elaeagnus umbellata Promotes Human Gingival Fibroblast Well-Being

Abstract

Four new triterpenoid bidesmosidic saponins (1-4) and a sesquiterpenoid glucoside (5), together with nine known phenolic compounds (6-14), were isolated from the fruits of Elaeagnus umbellata. Their structures were elucidated using 1D and 2D NMR spectroscopy and mass spectrometry data. The antioxidant capability of the isolated compounds was evaluated in human gingival fibroblasts. Compound 6 decreased ROS production and promoted cell proliferation. It also counteracted the cell cycle blockade induced by a low concentration of H₂O₂ decreasing the expression of p21 and CDKN2A (p16^INK4A). Compound 6 decreased the expression of inflammatory cytokines (IL-6 and IL-8) in response to inflammatory stimuli, supporting its possible use in periodontitis lesions.

Figure 1

Chemical profiles of Elaeagnus umbellata fruit extracts. (A) HPLC-ESIMS of the n-BuOH extract registered in the negative ion mode. (B) HPLC/UV of the EtOAc extract registered at 350 nm. Peak numbers indicate the isolated compounds.

Figure 2

Protective effects of Elaeagnus umbellata extracts and isolated compounds on hGF. Human GF cells were treated with different concentrations of Ex EtOAc, Ex BuOH, compounds 2, 5, and 6–14, quercetin (Q), kaempferol (K), or NAC (1 mM) in the absence (A) or presence (B) of H₂O₂ (200 μM) for 72 h in complete medium. In the end, the MTS assay was performed. Data are expressed as percentage of cell proliferation compared to the control, which was set to 100%. Each bar represents the mean ± SD of three replicates from two independent experiments. The significance of the differences was determined by one-way ANOVA, which was followed by Bonferroni’s post-test: *p < 0.05, **p < 0.01, ***p < 0.001, vs CTRL; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs H₂O₂.

Figure 3

Effect of compound 6 on ROS production, cell death, and cell cycle progression. hGF cells were treated with different concentrations of compound 6, quercetin (Q), kaempferol (K), or NAC as indicated, alone or in the presence of H₂O₂ (200 μM) for 72 h in complete medium. The ROS levels were evaluated by H₂DCFDA (A). Data are expressed as the percentage of ROS with respect to the amount in the CTRL set to 100%. Each bar represents the mean ± SD of three replicates from two independent experiments. (B) Flow cytometry analysis of cell death was performed as described in the Experimental Section. Data are reported as the number of cells/mL and as percentage of cell death. Each bar represents the mean ± SD of two replicates from two independent experiments. Representative histograms are shown. (C) Flow cytometry analysis of the cell cycle was performed as described in the Experimental Section. Data are reported as the percentage of cells in each cell phase. Each bar represents the mean ± SD of two replicates from two independent experiments. Representative histograms are shown. The significance of the differences was determined by one-way ANOVA, which was followed by Bonferroni’s post-test: *p < 0.05, **p < 0.01, ***p < 0.001, vs CTRL; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs H₂O₂.

Figure 4

Compound 6 modulation of senescence-associated and cytokine gene expression in hGF. hGF cells were treated with different concentrations of compound 6 alone or in the presence of H₂O₂ (200 μM) or LPS (1 μg/mL) for 72 h in complete medium. Then, the mRNA levels of p53 (A), p21 (B), p16^INK4A (C), IL-6 (D), IL-8 (E), and IL-10 (F) were quantified using real-time RT-PCR analysis. Data are expressed as the fold change versus the CTRL, which was set to 1, and are presented as the mean values ± SD of two independent experiments performed in duplicate. The significance of the differences was determined by one-way ANOVA, which was followed by Bonferroni’s post-test: *p < 0.05, **p < 0.01, ***p < 0.001 vs CTRL; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs H₂O₂ or LPS.