The Phenolic Profile and Anti-Inflammatory Effect of Ethanolic Extract of Polish Propolis on Activated Human Gingival Fibroblasts-1 Cell Line

Abstract

Propolis, owing to its antibacterial and anti-inflammatory properties, acts as a cariostatic agent, capable of preventing the accumulation of dental plaque and inhibiting inflammation. The anti-inflammatory properties of propolis are attributed to caffeic acid phenethyl ester (CAPE), which is present in European propolis. The objective of the conducted study was to assess the anti-inflammatory effects of the Polish ethanolic extract of propolis (EEP) and isolated CAPE on stimulated with LPS and IFN-α, as well as the combination of LPS and IFN-α. The cytotoxicity of the tested compounds was determined using the MTT assay. The concentrations of specific cytokines released by the HGF-1 cell line following treatment with EEP (25-50 µg/mL) or CAPE (25-50 µg/mL) were assessed in the culture supernatant. In the tested concentrations, both CAPE and EEP did not exert cytotoxic effects. Our results demonstrate that CAPE reduces TNF-α and IL-6 in contrast to EEP. Propolis seems effective in stimulating HGF-1 to release IL-6 and IL-8. A statistically significant difference was observed for IL-8 in HGF-1 stimulated by LPS+IFN-α and treated EEP at a concentration of 50 µg/mL (p = 0.021201). Moreover, we observed that CAPE demonstrates a stronger interaction with IL-8 compared to EEP, especially when CAPE was administered at a concentration of 50 µg/mL after LPS + IFN-α stimulation (p = 0.0005). Analysis of the phenolic profile performed by high-performance liquid chromatography allowed identification and quantification in the EEP sample of six phenolic acids, five flavonoids, and one aromatic ester-CAPE. Propolis and its compound-CAPE-exhibit immunomodulatory properties that influence the inflammatory process. Further studies may contribute to explaining the immunomodulatory action of EEP and CAPE and bring comprehensive conclusions.

Keywords: caffeic acid phenethyl ester; cytokines; in vitro model; inflammation; polyphenols; propolis.

Figure 1

Cell viability measured by MTT (%)—the cytotoxic activity of propolis ethanolic extract. The values represent the mean ± SD of three independent assays; * means p < 0.05 (calculated using t-test).

Figure 2

HGF-1 cells with CAPE incubated with MTT for 4 h presenting formazan crystals. (A). CAPE 10 µg/mL; (B). CAPE 25 µg/mL; (C). CAPE 50 µg/mL; (D). CAPE 100 µg/mL. (The arrow marks selected formazan crystals).

Figure 3

Cell viability measured by MTT (%)—the cytotoxic activity of CAPE. The values represent the mean ± SD of three independent assays; * means p < 0.05 (calculated using t-test).

Figure 4

Effect of EEP on selected cytokines production in native and stimulated HGF-1 by LPS, IFN-α, as well as combined LPS and IFN-α. (A). IL-1β, (B). IL-6, (C). IL-8, (D). IL-15, (E). TNF-α. The presented values mean ± SD of three independent experiments (n = 8); * means p < 0.05 (calculated using Fisher’s LSD Test).

Figure 4

Effect of EEP on selected cytokines production in native and stimulated HGF-1 by LPS, IFN-α, as well as combined LPS and IFN-α. (A). IL-1β, (B). IL-6, (C). IL-8, (D). IL-15, (E). TNF-α. The presented values mean ± SD of three independent experiments (n = 8); * means p < 0.05 (calculated using Fisher’s LSD Test).

Figure 4

Effect of EEP on selected cytokines production in native and stimulated HGF-1 by LPS, IFN-α, as well as combined LPS and IFN-α. (A). IL-1β, (B). IL-6, (C). IL-8, (D). IL-15, (E). TNF-α. The presented values mean ± SD of three independent experiments (n = 8); * means p < 0.05 (calculated using Fisher’s LSD Test).

Figure 5

Effect of CAPE on selected cytokines production in native and stimulated HGF-1 by LPS and IFN-α, as well as combined LPS and IFN-α; * means p < 0.05 (calculated using Fisher’s LSD Test).

Figure 5

Effect of CAPE on selected cytokines production in native and stimulated HGF-1 by LPS and IFN-α, as well as combined LPS and IFN-α; * means p < 0.05 (calculated using Fisher’s LSD Test).

Figure 5

Effect of CAPE on selected cytokines production in native and stimulated HGF-1 by LPS and IFN-α, as well as combined LPS and IFN-α; * means p < 0.05 (calculated using Fisher’s LSD Test).

Figure 6

Dendrogram obtained via the HCA analysis of all obtained data based on the average content of the effect of EEP and CAPE at different concentrations on pro-inflammatory cytokines.

Figure 7

Dendrogram obtained via the HCA analysis of all obtained data based on the influence on pro-inflammatory cytokines by the average content of the EEP and the CAPE at different concentrations.

Figure 8

PCA score plot of all obtained data based on the average content of the effect of EEP and CAPE at different concentrations on pro-inflammatory cytokines. PC—principal component.