Revisiting the Flora of Saudi Arabia: Phytochemical and Biological Investigation of the Endangered Plant Species Euphorbia saudiarabica

Abstract

Euphorbia plants have a significant place in traditional medicine due to their numerous therapeutic properties, including their anti-tumor effects, which have been observed in several species. In the current study, a phytochemical investigation of Euphorbia saudiarabica methanolic extract led to the isolation and characterization of four secondary metabolites from the chloroform (CHCl₃) and ethyl acetate (EtOAc) fractions, which are reported for the first time in this species. One of the constituents, saudiarabicain F (2), is a rare C-19 oxidized ingol-type diterpenoid that has not been previously reported. The structures of these compounds were determined by extensive spectroscopic (HR-ESI-MS, 1D and 2D NMR) analyses. The anticancer properties of the E. saudiarabica crude extract, its fractions and its isolated compounds were examined against several cancer cells. The active fractions were evaluated for their effects on cell-cycle progression and apoptosis induction using flow cytometry. Furthermore, RT-PCR was employed to estimate the gene-expression levels of the apoptosis-related genes. It was demonstrated that the E. saudiarabica CHCl₃ and EtOAc fractions suppressed the proliferation of the cancer cells. The MCF-7 cells were the most sensitive to both fractions, with IC₅₀ values of 22.6 and 23.2 µg/mL, respectively. Notably, both fractions caused cell-cycle arrest in the G2/M phase of the treated MCF-7 cells. The inhibition of the MCF-7 cells' proliferation was also linked with apoptosis induction by flow-cytometry analysis. Additionally, the activation of apoptosis by both fractions was demonstrated by an increase in the ratio of Bax to Bcl-2, with an increase in the expression of caspase-7. Among the isolated compounds, glutinol (1) showed potent activity against the MCF-7 cell line, with an IC₅₀ value of 9.83 µg/mL. Our findings suggest that E. saudiarabica has apoptosis-inducing effects and shows promise as a potential source of new chemotherapeutic drugs.

Keywords: Euphorbia saudiarabica; apoptosis; cytotoxicity; phytochemicals; saudiarabicain F.

Figure 1

Chemical structures of phytochemicals isolated from Euphorbia saudiarabica aerial parts.

Figure 2

COSY and HMBC correlations of saudiarabicain F (2) isolated from E. saudiarabica.

Figure 3

Cell viability of different cells following treatment with E. saudiarabica crude extract, CHCl₃ and EtOAc fractions. The cell viability was measured by MTT assay. Cells were treated with different concentrations of crude extract (A), CHCl₃ (B) or EtOAc (C) fractions. Data are presented as the mean ± standard deviation (SD) from three independent experiments.

Figure 4

Viability percentages of MCF-7 cells treated with compound 1. Cells were treated with the indicated concentration of compound 1 for 48 h and their viability was assessed by MTT assay. Results are expressed as Avg ± SD of three experiments.

Figure 5

Cell-cycle progression of MCF-7 cells after exposure to (A) CHCl₃ and (B) EtOAc fractions of E. saudiarabica. The MCF-7 cells were treated with 10- and 20-μg/mL concentrations for 24 h and the distribution of cells in various stages was evaluated by measuring DNA content. Results showed that both fractions induced G2/M cell-cycle arrest in a dose-dependent manner. The experiments were performed in triplicate. Data are reported as Avg ± SD. ** p ≤ 0.01 vs. control.

Figure 6

Effects of E. saudiarabica CHCl₃ and EtOAc fractions on stages of apoptosis. (A) The MCF-7 cells were incubated with 10 and 20 μg/mL of CHCl₃ fraction for 24 h and were then collected and stained with Annexin V/PI, followed by FACS analysis. The bars display FACS-quantification analysis. (B) The FACS and bar graphs showing the percentages of MCF-7 cells treated with 10 and 20 μg/mL of CHCl₃ fraction for 24 h. Data are mean ± SEM of three independent experiments, where * denotes p ≤ 0.05 and ** denotes p ≤ 0.01 vs. control, as measured by Student’s t-test.

Figure 7

Gene-expression analysis of Bcl-2, Bax and caspase-7 after incubation of MCF-7 cells with 10 and 20 μg/mL of (A) E. saudiarabica CHCl₃ fraction and (B) E. saudiarabica EtOAc fraction. Histograms showing densitometry analysis, in which band intensity was measured and normalized to that of β-actin. Data shown are means ± SD (n = 3) (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control).