Chemical, Antioxidant and Biological Studies of Brassica incana subsp. raimondoi (Brassicaceae) Leaf Extract

Abstract

Brassica incana subsp. raimondoi is an endemic taxon present in a restricted area located on steep limestone cliffs at an altitude of about 500 m a.s.l. in eastern Sicily. In this research, for the first time, studies on the phytochemical profile, the antioxidant properties in cell-free and cell-based systems, the cytotoxicity on normal and cancer cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, and on Artemia salina Leach, were performed. The total phenolic, flavonoid, and condensed tannin contents of the leaf hydroalcoholic extract were spectrophotometrically determined. Ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) analysis highlighted the presence of several phenolic acids, flavonoids, and carotenoids, while High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) identified various kaempferol and isorhamnetin derivatives. The extract exhibited different antioxidant properties according to the five in vitro methods used. Cytotoxicity by MTT assay evidenced no impact on normal human fibroblasts (HFF-1) and prostate cancer cells (DU145), and cytotoxicity accompanied by necrotic cell death for colon cancer cells (CaCo-2) and hepatoma cells (HepG2), starting from 100 μg/mL and 500 μg/mL, respectively. No cytotoxic effects were detected by the A. salina lethality bioassay. In the H₂O₂-induced oxidative stress cell model, the extract counteracted cellular reactive oxygen species (ROS) production and preserved non-protein thiol groups (RSH) affected by H₂O₂ exposure in HepG2 cells. Results suggest the potential of B. incana subsp. raimondoi as a source of bioactive molecules.

Keywords: Artemia salina Leach; H2O2; HPLC/DAD; ROS; UPLC-MS/MS; botanicals; cabbages; carotenoids; nutraceuticals; oxidative stress; polyphenols.

Figure 1

Brassica incana subsp. raimondoi (Sciandr., C. Brullo, Brullo, Giusso, Miniss. and Salmeri) Raimondo and Spadaro in the respective locus classicus (Castelmola, Messina, Italy).

Figure 2

HPLC-DAD polyphenols fingerprint of Brassica raimondoi leaf extract (mAU—milli-absorbance unit). Column: Ascentis Express C18, 15 cm × 4.6 mm, 2.7 µm d.p. The numbers indicating peaks refer to the identified compounds reported in Table 3: kaempferol-3-O-diglucoside-7-O-glucoside (1); kaempferol-3-hdroxyferuloylsophoroside-7-glucoside (2); quercetin-3-feruloyl-diglucoside-7-glucoside (3); isorhamnetin-3-glucoside-7-glucoside (4); kaempferol-rutinoside (5).

Figure 3

Cell viability in HFF-1, HepG2, DU145, and CaCo-2 cells untreated (Ctr), and treated with Brassica raimondoi extract for 24 h (a). Cell viability in HepG2 cells untreated (Ctr), treated for 2 h with H₂O₂ 200 μM (H₂O₂), and pre-treated with the extract (10–50–100–200–400 μg/mL) followed by H₂O₂ treatment (200 μM) for 2 h (b). Values are the mean ± S.D. of five experiments in triplicate. Confidence intervals calculated by two-way ANOVA test: * Significant vs. untreated control cells: p < 0.001; # Significant vs. H₂O₂ treated cells: p < 0.001.

Figure 4

LDH release in untreated HepG2 and CaCo-2 cells (Ctr) and treated for 24 h with the extract (10–1000 μg/mL). Values are the mean ± S.D. of five experiments in triplicate. Confidence intervals calculated by two-way ANOVA test: * Significant vs. untreated control cells: p < 0.001.

Figure 5

ROS production in HepG2 untreated cells (Ctr), treated for 2 h with H₂O₂ 200 μM (H₂O₂), and pre-treated with Brassica raimondoi extract (10–50–100–200–400 μg/mL) and followed by H₂O₂ treatment 200 μM 2 h. Results are expressed as percentage of fluorescence intensity (I.F.)/mg protein. Values are the mean ± S.D. of five experiments in triplicate. Confidence intervals calculated by two-way ANOVA test: ^* Significant vs. untreated control cells: p < 0.001; # Significant vs. H₂O₂ treated cells: p < 0.001. I.F. vs. H₂O₂.: Intensity of Fluorescence vs. H₂O₂ treated cells.

Figure 6

RSH (non-protein thiol groups) levels in HepG2 untreated cells (Ctr), treated for 2 h with H₂O₂ 200 μM (H₂O₂), and pre-treated with Brassica raimondoi extract (10–50–100–200–400 μg/mL) and followed by H₂O₂ treatment 200 μM 2 h. Values are the mean ± S.D. of five experiments in triplicate. Confidence intervals calculated by two-way ANOVA test: ^* Significant vs. untreated control cells: p < 0.001; # Significant vs. H₂O₂ treated cells: p < 0.001. % RSH levels vs Ctr.: percentage of non-protein thiol groups vs. untreated control cells.

Figure 7

Leaf characteristics and morphology of Brassica incana subsp. raimondoi (Sciandr., C. Brullo, Brullo, Giusso, Miniss. and Salmeri) used in this research.