Polypodium vulgare L. (Polypodiaceae) as a Source of Bioactive Compounds: Polyphenolic Profile, Cytotoxicity and Cytoprotective Properties in Different Cell Lines

Abstract

Pteridophytes, represented by ferns and allies, are an important phytogenetic bridge between lower and higher plants. Ferns have evolved independently of any other species in the plant kingdom being its secondary metabolism a reservoir of phytochemicals characteristic of this taxon. The study of the potential uses of Polypodium vulgare L. (Polypodiaceae) as medicinal plant has increased in recent years particularly when in 2008 the European Medicines Agency published a monograph about the rhizome of this species. Our objective is to provide scientific knowledge on the polar constituents extracted from the fronds of P. vulgare, one of the main ferns of European distribution, to contribute to the validation of certain traditional uses. Specifically, we have characterized the methanolic extract of P. vulgare fronds (PVM) by HPLC-DAD and investigated its potential cytotoxicity, phototoxicity, ROS production and protective effects against oxidative stress by using in vitro methods. The 3T3, HaCaT, HeLa, HepG2, MCF-7 and A549 were the cell lines used to evaluate the possible cytotoxic behaviour of the PVM. HPLC-DAD was utilized to validate the polyphenolic profile of the extract. H₂O₂ and UVA were the prooxidant agents to induce oxidative stress by different conditions in 3T3 and HaCaT cell lines. Antioxidant activity of in vitro PVM in 3T3 and HaCaT cell lines was evaluated by ROS assay. Our results demonstrate that PVM contains significant amounts of shikimic acid together with caffeoylquinic acid derivatives and flavonoids such as epicatechin and catechin; PVM is not cytotoxic at physiological concentrations against the different cell lines, showing cytoprotective and cellular repair activity in 3T3 fibroblast cells. This biological activity could be attributed to the high content of polyphenolic compounds. The fronds of the P. vulgare are a source of polyphenolic compounds, which can be responsible for certain traditional uses like wound healing properties. In the present work, fronds of the common polypody are positioned as a candidate for pharmaceutical applications based on traditional medicine uses but also as potential food ingredients due to lack of toxicity at physiological concentrations.

Keywords: cytoprotection; cytotoxicity; ferns; medicinal plants; polyphenols; polypody.

IMAGE 1

Photography of the face fronds (A) and underside frond (B) of fresh Polypodium vulgare L. (Polypodiaceae). Pictures were taken by Adrià Farràs at Prades mountains. The euro coin reflects the dimension of the frond (image 1B).

FIGURE 1

HPLC-DAD chromatograms reported only at 272 nm for sake of clarity and corresponding to (A) standard mixture solution (B) extract of methanolic fronds extract of Polypodium vulgare L. List of compounds: 1 = shikimic acid, 2 = gallic acid, 3 = 5-O-caffeoylquinic acid, 4 = 3-O-caffeoylquinic acid, 5 = catechin, 6 = epicatechin, 7 = rutin, 8 = hyperoside, 9 = 3,5-di-O-caffeoylquinic acid.

FIGURE 2

Cytotoxicity activity of PVM in 3T3, HaCaT, HeLa, HepG2, MCF-7 and A549 cell lines by MTT assay and expressed as percentage of cell viability respect to control cells. Results are expressed as mean ± standard error of n = 3. Control cells were maintained only with 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 compared with no treated cells (negative control).

FIGURE 3

Cytoprotective activity of PVM in 3T3 and HaCaT cell lines for 2 mM H₂O₂ during 2.5 h by MTT assay and expressed as percentage of cell viability respect to untreated cells control. H₂O₂ cell viability was used as positive control. Results are expressed as mean ± standard error of n = 3 and n = 2 respectively. A two-way analysis of variance (ANOVA) and a Bonferroni post hoc assay have been performed. No statistically significant differences were found.

FIGURE 4

Cellular repair activity of PVM in 3T3 cell line for 2 mM H₂O₂ during 2.5 h by MTT assay and expressed as percentage of cell viability respect to untreated cells control. H₂O₂ cell viability was used as 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 ≤ 0.001 and ****p ≤ 0.0001 compared with positive control.

FIGURE 5

Phototoxicity activity of PVM in 3T3 and HaCaT cell lines by MTT assay and expressed as percentage of cell viability respect to the correspondent control cells. Chloropromazine cell viability was used as positive control. Gray columns correspond to cells non exposed 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 ≤ 0.01 and ****p ≤ 0.0001 compared with correspondence no irradiated/irradiated positive control.

FIGURE 6

Intracellular ROS induced by 1 and 2 mM H₂O₂ for 2 h treatment with PVM in 3T3 and HaCaT cells. H₂O₂: 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, **p ≤ 0.01 and ***p ≤ 0.001 compared with the correspondent positive control.