Chemical Characterization, Free Radical Scavenging, and Cellular Antioxidant Properties of the Egadi Island Endemic Brassica macrocarpa Guss Leaf Extract

Abstract

The genus Brassica is an important source of food in the Mediterranean diet with documented nutritional and medicinal properties. However, few studies have investigated the phytochemical composition and the biological activity of wild Sicilian taxa. Thus, we aimed to study the chemical profile and the antioxidant potential, in vitro and in LPS-stimulated RAW 264.7 cells, of a methanolic extract of leaves of wild Brassica macrocarpa Guss (B. macrocarpa) (Egadi Islands; Sicily-Italy). B. macrocarpa methanolic extract showed a large amount of glucosinolates and different phenolic compounds. It exhibited antioxidant activity in the DPPH assay and in LPS-stimulated RAW 264.7 cells, being able to reduce NO and ROS levels and NOS2 mRNA expression. Our study demonstrated that Sicilian B. macrocarpa methanolic extract, in LPS-stimulated macrophages, efficiently counteracts oxidative stress and displays radical scavenging activity. Future studies are required to identify the contribution of the single phytocomponents, to characterize the action mechanism, and to reveal possible applications in human health.

Keywords: Brassica macrocarpa; Brassicaceae; ROS; antioxidant activity; scavenging activity.

Figure 1

LC-MS chromatogram (BPI) of B. macrocarpa methanolic extract.

Figure 2

Antioxidant capacity of B. macrocarpa methanolic extract measured as DPPH scavenging capacity. Data are expressed as mean ± SEM (n = 3). * p < 0.05.

Figure 3

Effect of B. macrocarpa methanolic extract on the viability of RAW 264.7 cells. Cells were treated for 24 h with extract at the concentration range from 7.81 to 1000 µg/mL, and cell viability was assessed by MTT assay. Data are mean ± SEM and expressed as the percentage of control cells.

Figure 4

Effect of different doses of LPS on the viability of RAW 264.7 cells. Cells were treated for 24 h with LPS at the concentration range from 0.1 to 1 µg/mL, and cell viability was assessed by MTT assay. Data are mean ± SEM (n = 3) and expressed as the percentage of control cells. * p < 0.05.

Figure 5

Effects of LPS on nitric oxide production in RAW 264.7 cells at different time points. The amount of nitric oxide produced was determined by Griess assay. Data are expressed as mean ± SEM (n = 3). * p < 0.05 compared to the untreated cells (time 0).

Figure 6

Effect of the joint application of LPS (0.1 μg/mL) and B. macrocarpa methanolic extract (concentration range from 7.81 to 1000 µg/mL) on the viability of RAW 264.7 cells. Cell viability was assessed by MTT assay, and no toxicity was observed. Data are mean ± SEM (n = 3) and expressed as the percentage of control cells.

Figure 7

Effects of B. macrocarpa methanolic extract on nitric oxide production in LPS-stimulated RAW 264.7 cells. The amount of nitric oxide produced was determined by Griess assay. Data are expressed as mean ± SEM (n = 3). * p < 0.05 compared to the cells treated with LPS alone (LPS group).

Figure 8

Effects of B. macrocarpa methanolic extract on the relative mRNA expression levels of NOS2 in LPS-stimulated RAW 264.7 cells. The cells were treated with the extract for 2 h and then stimulated with LPS (0.1 μg/mL). The mRNA levels were measured by qRT-PCR. Data are expressed as mean ± SEM (n = 3). * p < 0.05 compared to the cells treated with LPS alone (LPS group).

Figure 9

Effects of B. macrocarpa methanolic extract on ROS production in LPS-stimulated RAW 264.7 cells. The amount of ROS produced was determined using the H₂DCF-DA assay. Data are expressed as mean ± SEM (n = 3). * p < 0.05 compared to the cells treated with LPS alone (LPS group).