Exploring wild Aspleniaceae ferns as safety sources of polyphenols: The case of Asplenium trichomanes L. and Ceterach officinarum Willd

Abstract

The forest ecosystem is a source of material resources used since ancient times by mankind. Ferns are part of different oriental systems of traditional medicine due to the phytochemical variety of their fronds, which have allowed their traditional use to be validated through ethnopharmacological studies. In Europe, different cultures have used the same fern with a wide variety of applications due to its presence in most European forests. In recent years, studies on the phytocharacterization and biological activity of the fronds of the main European ferns have been published. In this study, the presence of polyphenolic phytochemicals has been evaluated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the fronds of two wild ferns together with in vitro activities in non-tumoral and human tumoral cell lines. The polyphenols were extracted from Asplenium trichomanes L. and Ceterach officinarum Willd. by cold maceration using methanol. The main phytochemicals of polyphenolic origin in the extracts of A. trichomanes and C. officinarum determined by HPLC-MS/MS were the flavonol hyperoside and the phenolic acid chlorogenic acid, respectively. This different polyphenolic nature of both extracts contributes to the divergence of the behavior experienced in the biological activities tested, but none of the extracts showed a cytotoxic or phototoxic profile in the different tested cell lines. However, the cytoprotective values in front of the H₂O₂ oxidative stress induced in the 3T3 and HaCaT cell lines position these extracts as possible candidates for future health applications.

Keywords: bioeconomy; cytoprotection; cytotoxicty; ethnopharmacology; ferns; functional food; polyphenolic phytochemicals.

IMAGE 1

Face fronds (1) and underside frond (2) of fresh Asplenium trichomanes L. (Aspleniaceae) (A) and fresh Ceterach officinarum Willd. (Aspleniaceae) (B) by Adrià Farràs at Prades mountains. The euro coin referents the dimension of the frond (A2, B2).

FIGURE 1

Cytotoxicity activity of ATM (1) and COM (2) in 3T3 (A) and HaCaT (B), HeLa (C), HepG2 (D), MCF-7 (E), and A549 (F) cell lines by MTT assay and expressed as a percentage of cell viability respect to control cells Results are expressed as mean ± standard error of n = 3. Control cells were maintained only with a culture medium. A two-way analysis of variance (ANOVA) and a Bonferroni post hoc assay have been performed. Statistical differences were considered as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 comparison with no treated cells (negative control).

FIGURE 2

Cytoprotective activity of ATM (1) and COM (2) in 3T3 (A) and HaCaT (B) cell lines for 2 mM H₂O₂ during 2.5 h by MTT assay and expressed as a percentage of cell viability with respect to untreated cells control. H₂O₂ cell viability was used as a positive control. Results are expressed as mean ± standard error of n = 3 (3T3) and n = 2 (HaCaT). A two-way analysis of variance (ANOVA) and a Bonferroni post hoc assay have been performed. Statistical differences were considered as follows: ****p ≤ 0.0001 comparison with positive control.

FIGURE 3

Cellular repair activity of ATM (A) and COM (B) in 3T3 cell line for 2 mM H₂O₂ during 2.5 h by MTT assay and expressed as a percentage of cell viability with respect to untreated cells control. H₂O₂ cell viability was used as a positive control. Results are expressed as mean ± standard error of n = 3. A two-way analysis of variance (ANOVA) and a Bonferroni post hoc assay have been performed. Statistical differences were considered as follows: *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001 comparison with positive control.

FIGURE 4

Phototoxicity activity of ATM (1) and COM (2) in 3T3 (A) and HaCaT (B) cell lines by MTT assay and expressed as a percentage of cell viability with respect to the correspondent control cells Chlorpromazine cell viability was used as positive control. Gray columns correspond to cells nonexposed to UVA light and white columns correspond to cells exposed to 1.8 J/cm² of UVA light. Results are expressed as mean ± standard error of n = 3. A two-way analysis of variance (ANOVA) and a Bonferroni post hoc assay have been performed. Statistical differences were considered as follows: *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001 comparison with the equivalent nonirradiated condition homologue.

FIGURE 5

Intracellular ROS induced by 1 and 2 mM H₂O₂ for 2 h treatment with ATM (1) and COM (2) in 3T3 (A) and HaCaT (B) cells H₂O₂ cell viability was used as a positive control. White columns correspond to 1 mM H₂O₂, and gray columns correspond to 2 mM H₂O₂. Results are expressed as mean ± standard error of n = 3. A two-way analysis of variance (ANOVA) and a Bonferroni post hoc assay have been performed. Statistical differences were considered as follows: *p ≤ 0.05 and **p ≤ 0.01 comparison with the corresponding positive control.