Ferula communis L. (Apiaceae) Root Acetone-Water Extract: Phytochemical Analysis, Cytotoxicity and In Vitro Evaluation of Estrogenic Properties

Abstract

Ferula communis L. (F. communis) belongs to the Apiaceae family and is a herbaceous plant with various pharmaceutical properties, due to the different contents of bioactive compounds extracted mainly from its roots, as well as its leaves and rhizome. To date, this plant extract has demonstrated estrogenic, anti-inflammatory, antiproliferative, cytotoxic, antimicrobial and anti-neoplastic properties. Its estrogenic activity is justified by the presence of ferutinin, an ester of a sesquiterpenic alcohol that acts as an agonist for estrogen receptors, with a chemical formula equal to C₂₂H₃O₄. The component present in F. communis responsible for the toxicity of the plant is ferulenol, a prenylated coumarin with the chemical formula C₂₄H₃₀O₃. This compound is capable of inducing mortality via its strong anti-coagulant properties, leading to a lethal hemorrhagic syndrome, ferulosis, in animals that feed on a chemotype of F. communis containing a high amount of ferulenol. The removal of the component ferulenol makes extracts of Ferula non-toxic. In fact, the remaining prenylated coumarins are not present in concentrations sufficient to induce toxicity. The intake of high concentrations of the extract of this plant leads a double dose-dependent effect that is typical of sesquiterpenes such as ferutinin. Here, we assessed the cytotoxicity and the estrogenic properties of the F. communis phytocomplex obtained through extraction using a mixture of acetone and water. Among the active constituents of F. communis, the identification of ferutinin and ferulenol was performed using HPLC. The effects of the extract were evaluated, following the removal of ferulenol, on three cell lines: human breast cancer MCF-7, human cervical cancer HeLa and human osteoblastic sarcoma Saos-2. The choice of these cell lines was justified by the need to mimic certain processes which may occur in vivo and which are estrogen-dependent. The obtained results demonstrated that F. communis extract, in addition to possessing an estrogenic-like property, showed a dose-dependent effect. Low concentrations (0.1-0.8 μM) demonstrated a hyperproliferative effect, whereas higher concentrations (1.6-50 μM) were toxic. Therefore, this extract could be an excellent candidate to make up for a reduction or lack of estrogen.

Keywords: Ferula communis L. extract; HeLa cells; MCF-7 cells; Saos-2 cells; ferulenol; ferutinin.

Figure 1

In panel (a) the chemical structures of ferutinin and ferulenol are represented; in panel (b) HPLC spectrum of FER-E, in which ferutinin and ferulenol are present, is shown. In panel (c) HPLC analysis values are represented.

Figure 2

HPLC spectrum of FER-E in which ferulenol is absent. In panel (a) the HPLC spectrum of FER-E is shown. In panel (b), HPLC analysis values are represented.

Figure 3

Hyperproliferation induced by treatment with 17-β-E2 in MCF-7, HeLa and Saos-2 cell lines. Cell lines were treated with increasing concentrations of 17-β-E2 (0.01–10 μM) and the proliferation rate was measured through the MTT test. Each panel is composed of three curves obtained from the data measured at 24, 48 and 72 h of treatment. In particular, panel (a) shows the results related to the cell line MCF-7; panel (b) refers to HeLa cells; and finally, panel (c) highlights the data related to Saos-2 cells. Three independent experiments were carried out, and the values are expressed as the mean ± standard deviation (sd). * denotes p < 0.05 versus the control; ** denotes p < 0.01 versus the control; *** denotes p < 0.001 versus the control. Analysis of Variance (ANOVA) was followed by a Tukey–Kramer comparison test. Each treatment was carried out in quadruplicate.

Figure 4

Dose-dependent effect induced by FER-E treatment. Dose–response curves were constructed by treating the cell lines considered with increasing concentrations of FER-E. Panels (a–c) refer to viability data induced in MCF-7, HeLa e Saos-2, respectively. Three independent experiments were carried out and the values are expressed as the mean ± standard deviation (sd). * denotes p < 0.05 versus the control; ** denotes p < 0.01 versus the control; *** denotes p < 0.001 versus the control. Analysis of Variance (ANOVA) was followed by a Tukey–Kramer comparison test. Each treatment was carried out in eightfold.

Figure 5

Percentage of mortality induced by treatment with FER-E. Percentage of mortality was calculated following treatment with FER-E for 24, 48 and 72 h by the Trypan blue test. In particular, mortality induced by treatment with FER-E on MCF-7, HeLa and Saos-2 cell lines was shown in panels (a–c), respectively. Three independent experiments were carried out, and the values are expressed as the mean ± standard deviation (sd). * denotes p < 0.05 versus the control; ** denotes p < 0.01 versus the control; *** denotes p < 0.001 versus the control. Analysis of Variance (ANOVA) was followed by a Tukey–Kramer comparison test. Each treatment was carried out in triplicate.

Figure 6

Mortality assessed by Annexin V Staining assay. Figure 6 shows the effect of treatment with different concentrations of FER-E on cell lines. In particular, in panels (a–c) the results for lines MCF-7, HeLa and Saos-2 are represented, respectively. Cytometric analysis was conducted on 20,000 events and each treatment is represented by a dot plot divided into 4 quadrants (Q1, Q2, Q3, and Q4). Q1 refers to Annexin V-negative/PI-negative cells (viable cells). Q2 refers to Annexin V-positive/PI-negative cells (apoptotic cells). Q3 refers to Annexin V-positive/PI-positive cells (late apoptosis). Q4 refers to Annexin V-negative/PI-positive cells (advanced necrosis). A representative experiment of three independent experiments was shown.

Figure 7

Expression of results obtained by Annexin V Staining assay. The results of three independent experiments, obtained by Annexin V Staining assay are shown. In particular, the percentage of cellular fluorescence in the total number of events (20,000) has been evaluated and indicated in the relevant box. In panels (a–c) the quantifications of MCF7, HeLa and Saos-2 cell lines are shown respectively. Data from an indicative experiment (of 3 independent experiments with similar results) are shown.