Isolation, NMR Characterization, and Bioactivity of a Flavonoid Triglycoside from Anthyllis henoniana Stems: Antioxidant and Antiproliferative Effects on MDA-MB-231 Breast Cancer Cells

Abstract

Plant extracts are considered as a large source of active biomolecules, especially in phytosanitary and pharmacological fields. Anthyllis henoniana is a woody Saharan plant located in the big desert of North Africa. Our previous research paper proved the richness of the methanol extract obtained from the stems in flavonoids and phenolic compounds as well as its remarkable antioxidant activity. In this research, we started by investigating the phytochemical composition of the methanol extract using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS/MS). Among the 41 compounds identified, we isolated and characterized (structurally and functionally) the most abundant product, a flavonoid triglycoside (AA₇₇₀) not previously described in this species. This compound, which presents no cytotoxic activity, exhibits an interesting cellular antioxidant effect by reducing reactive oxygen species (ROS) generation, and an antiproliferative action on breast cancer cells. This study provides a preliminary investigation into the pharmacological potential of the natural compound AA₇₇₀, isolated and identified from Anthyllis henoniana for the first time.

Keywords: Anthyllis henoniana; LC–MS/MS; ROS; cell proliferation; cell viability; flavonoids NMR; isorhamnetin-3-O-rutinoside-7-O-rhamnopyranoside; phenolic compounds.

Figure 1

Chromatograms of Anthyllis henoniana methanol extract. (a): Mass chromatogram. (b): UV–DAD chromatogram. (c): Absorption of AA₇₇₀ compound (retention time = 20.95 min).

Figure 2

ESI–MS mass spectra of compound 17(AA₇₇₀): Isorhamnetin-3-O-rutinoside-7-O-rhamnopyranoside: (a) full scan mass spectrum; (b): MS² of 769, giving m/z = 623, corresponding to the loss of rhamnose; (c): MS³ of 769, fragmentation of 623.17 mainly generated the aglycone m/z = 315.

Figure 3

Structure of AA₇₇₀: Isorhamnetin-3-O-rutinoside-7-O-rhamnopyranoside.

Figure 4

Effect of AA₇₇₀ on MDA-MB-231 cytotoxicity and viability. Cytotoxicity of AA₇₇₀ was tested by measuring cell mortality by LDH release (A) and viability by CCK-8 assay (B). Results are expressed as percentages with respect to the positive control for each assay: lysis buffer for LDH and DMSO for CCK8. The values are means ± SE of four independent experiments, each performed in triplicate. For the LDH assay, positive control is obtained with lysis buffer. In both assays, DMSO conditions were established by incubating the samples with 0.5% DMSO for 24 h. Means were statistically compared to DMSO (ns: not significant, *** p < 0.001 Tukey’one-way ANOVA test).

Figure 5

Evaluation of ROS production in non-treated MDA-MB-231 cells and cells treated with TBHP, AA₇₇₀, and isorhamnetin (IsoR). Cells were incubated for 4 h with buffer (Basal, black bar) or buffer with TBHP at 30 µmol·L⁻¹ plus 0.5% DMSO (white bar), 3 mmol·L⁻¹ NAC (grey bar), or various concentrations of AA₇₇₀ (red bar) or isorhamnetin (blue bar) as indicated. After incubation, ROS levels were evaluated using the 2′,7′-dichlorodihydrofluorescein diacetate assay. Histograms indicate the normalized fluorescence for each condition using TBHP as reference. The bars represent the means ± SE of three independent experiments, each performed in triplicate. Means were found statistically different compared to TBHP (** p < 0.01; *** p < 0.001 Tukey’s one-way ANOVA test).

Figure 6

Effects of AA₇₇₀ and isorhamnetin on MDA-MB-231 cells proliferation. MDA-MB-231 cells were treated daily with DMSO or 10 µmol·L⁻¹ of isorhamnetin or AA₇₇₀. Cell proliferation was measured using CCK-8 assay every day for 4 days after the beginning of the treatment. Results are presented as relative absorbance at 450 nM (mean ± SE, n = 3, each performed in triplicate).