Effect of Punicalagin and Ellagic Acid on Human Fibroblasts In Vitro: A Preliminary Evaluation of Their Therapeutic Potential

Abstract

Background: Pomegranate is a fruit that contains various phenolic compounds, including punicalagin and ellagic acid, which have been attributed to anti-inflammatory, antioxidant, and anticarcinogenic properties, among others.

Objective: To evaluate the effect of punicalagin and ellagic acid on the viability, migration, cell cycle, and antigenic profile of cultured human fibroblasts (CCD-1064Sk). MTT spectrophotometry was carried out to determine cell viability, cell culture inserts were used for migration trials, and flow cytometry was performed for antigenic profile and cell cycle analyses. Cells were treated with each phenolic compound for 24 h at doses of 10^-5 to 10^-9 M.

Results: Cell viability was always significantly higher in treated versus control cells except for punicalagin at 10^-9 M. Doses of punicalagin and ellagic acid in subsequent assays were 10^-6 M or 10^-7 M, which increased the cell migration capacity and upregulated fibronectin and α-actin expression without altering the cell cycle.

Conclusions: These in vitro findings indicate that punicalagin and ellagic acid promote fibroblast functions that are involved in epithelial tissue healing.

Keywords: ellagic acid; fibroblasts; phenolic compounds; pomegranate; punicalagin.

Figure 1

Effects of punicalagin (a) and ellagic acid (b) on the cell viability of fibroblasts at 24 h of treatment. EC₅₀ value for each treatment (c,d). * p < 0.05. It is observed that treatment with punicalagin at doses of 10⁻⁵ to 10⁻⁸ M and ellagic acid at all concentrations significantly increase cell viability.

Figure 2

Percentages of cells in G0-G1, G2-M, and S phase at 24 h of treatment with punicalagin and ellagic acid as measured by flow cytometry. Significant alterations in the percentage of cells within each phase of the cell cycle are not observed. AE: ellagic acid; Pun: punicalagin.

Figure 3

(A). Effect of punicalagin and ellagic acid on the migratory capacity of human fibroblasts. Punicalagin at 10⁻⁷ M and ellagic acid at 10⁻⁶ M significantly increased their capacity at 4 (p < 0.003 and p < 0.001), 8 (p < 0.005 and p < 0.001), 12 (p < 0.001 and p < 0.001), and 24 h (p < 0.001 and p < 0.001). Punicalagin at 10⁻⁶ M significantly increased it at 24 h (p < 0.001), and ellagic acid at 10⁻⁶ M significantly increased it at 4 (p < 0.003), 12 (p < 0.002), and 24 h (p < 0.001). AE: ellagic acid; Pun: punicalagin. (B). Bright-field microscope images of each treatment at 24 h. An observed reduction in the percentage of gap closure is evident across all treatments at varying doses compared to control cells. AE: ellagic acid; Pun: punicalagin.

Figure 4

Expression of α-actin and fibronectin by cultured human fibroblasts at 24 h of treatment with punicalagin and ellagic acid. The percentage expression of α-actin and fibronectin was significantly higher in cells treated with different compounds at all selected doses. AE: ellagic acid; Pun: punicalagin. * p ≤ 0.05.

Figure 5

Immunostaining of human fibroblast cells where increased labeling of fibronectin-fluorescein (green) and α-actin-phycoerythrin (red) is observed in cells treated with punicalagin (Pun) and ellagic acid (AE) at the different doses tested at 24 h of treatment. The blue color in the nucleus shows DAPI staining.