Antifungal and Cytotoxic Activity of Diterpenes and Bisnorsesquiterpenoides from the Latex of Euphorbia resinifera Berg

Abstract

Euphorbia resinifera latex has been extensively utilized in traditional medicine due to its range of bioactivities. Chromatographic separations on silica gel of ethanol extract of E. resinifera latex led to the development of a new procedure for isolating resiniferatoxin (4) via dried E. resinifera latex and the identification of nine compounds. Among these, catechol (7), protocatechuic acid (8) and 3,4-dihydroxyphenylacetic acid (9), known phenolic compounds, were identified for the first time in E. resinifera latex. Herein we investigated the effects of major compounds of the latex of E. resinifera on the yeast Saccharomyces cerevisiae, on the growth of Aspergillus carbonarius, a widespread fungal contaminant, and on the breast cancer cell line MCF7 as well as on MCF10A normal breast cells. 12-deoxyphorbol-13-isobutyrate-20-acetate (2) had an inhibiting effect on the growth of A. carbonarius, and 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) showed a negative effect on yeast cell growth and also a cytotoxic effect on breast cancer cell line MCF7, but not on MCF10A cells. Deglucosyl euphorbioside A (5) and euphorbioside A (6) showed a discoloration effect that was possibly related to mitochondrial functionality in yeast, and also cytotoxicity only on the cancer cell line that was tested. Interestingly, treatment of MCF7 cells with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) not only led to a specific cytotoxic effect but also to the increase in the level of intracellular ROS.

Keywords: Aspergillus carbonarius; Euphorbia resinifera; Saccharomyces cerevisiae; biological effect; bisnorsesquiterpenoids; breast cancer cell line; diterpenes.

Figure 1

Chemical structure of compounds 1–9 that were isolated from the latex of Euphorbia resinifera.

Figure 2

Effects of the tested compounds on S. cerevisiae W303 exponential growth phase. (A): yeast cells that were treated with compounds at 400 μM; 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) showed a negative effect on yeast cell growth, while 12-deoxyphorbol-13-angelate-20-acetate (1) had a positive effect. (B): Cells that were treated with compound 3 showed a bigger size with swollen vacuoles. The experiment was conducted in three independent replicates.

Figure 3

H₂O₂ sensitivity of S. cerevisiae that was induced by the tested compounds. The sensitivity was visualized with a growth inhibition test. Deglucosyl euphorbioside A (5) and euphorbioside A (6) induced a slight sensitivity to H₂O₂ producing a halo of 3.7 cm and 4 cm, respectively, bigger than the halo that was produced with cells that were treated with DMSO (3 cm).

Figure 4

The effect of 12-deoxyphorbol-13-isobutyrate-20-acetate (2) on the growth of Aspergillus carbonarius. The fungus was cultured in CDY 0.2% at 25 °C. The data represent the average with standard deviations from three independent experiments. The results are expressed as percentages of growth normalized to untreated fungal culture (CON).

Figure 5

Comparison of the cell viability of MCF10A and MCF7 cell lines that were treated for 72 h with different concentrations of compounds (0.001, 1, 10, and 100 μΜ). Cell viability was measured by MTS assay. The line-graphs represent the average with standard deviations from three independent experiments. Results are expressed as the percentage of cell viability normalized to the untreated cells. Statistical differences between MCF7 and MCF10A cells were assessed by Student’s t-test (* p < 0.05, *** p < 0.001), ns = not significant.

Figure 6

Analysis of mitochondrial ROS using the dye MitoSox Red in cells that were incubated 1 h with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) at 10 μM. The data are represented with fold of induction compared to the untreated samples (relative value). All the values are expressed as the average with standard deviations from three independent experiments. Statistical differences between MCF7 and MCF10A cells that were treated at 10 µM were assessed by Student’s t-test (*** p < 0.001).