Antioxidant and Anti-Inflammatory Effects of Extracts from Pulsed Electric Field-Treated Artichoke By-Products in Lipopolysaccharide-Stimulated Human THP-1 Macrophages

Abstract

In this study, pulsed electric field (PEF-3 kV/cm; 5 kJ/kg) pretreatment was used to intensify the extractability of valuable intracellular compounds from artichoke by-products during a subsequent aqueous extraction (solid-liquid ratio = 1:10 g/mL, T = 20 °C; t = 120 min). Total phenolic content (TPC), antioxidant activity (DPPH, ABTS) and HPLC-PDA analysis of the artichoke extract (AE) and the biological effects on human cell lines were determined. Chlorogenic acid was found to be the most abundant phenolic compound (53% of the TPC) in the AE. The extract showed good antioxidant properties in a concentration-dependent manner. The potential biological effects of AE were investigated using THP-1 macrophages stimulated by lipopolysaccharides (LPS) as an in vitro model system of oxidative stress. Reduced reactive oxygen species production upon treatment with AE was found. Moreover, AE was able to reduce the secretion of the pro-inflammatory mediators Interleukin-6 and Monocyte Chemoattractant Protein-1 in LPS-stimulated macrophages, as determined by qRT-PCR and ELISA assays. These results highlighted the anti-inflammatory and antioxidant properties of the extracts from PEF-treated artichoke by-products, corroborating their potential application as a source of functional ingredients obtained through a feasible and sustainable process.

Keywords: HPLC–PDA; PEF-assisted extraction; artichoke residues; bioactivity; human cell lines; phenolic compounds.

Figure 1

HPLC–PDA chromatograms of the extract from untreated artichoke stems (black line), and the extract from PEF-treated artichoke stems (red line). Peak identification: gallic acid (1), catechin (2), chlorogenic acid (3), epicatechin (4), cynarine (5), sinapic acid (6), naringin (7), rutin (8), phlorizin (9).

Figure 2

Changes in %I_DPPH and %I_ABTS of AE at different concentrations. Extract I% vs. initial concentration of ABTS or DPPH (0). The values represent the means ± SDs of three different experiments, each performed in triplicate. n.s.: not significant; * p < 0.05; **** p < 0.0001.

Figure 3

Reactive oxygen species production in lipopolysaccharide-stimulated human macrophages upon treatment with artichoke extract. (A) Representative fluorescent microscopy images of reactive oxygen species (ROS) detection in THP-1 macrophages untreated (−) or treated with 1 and 2 mg/mL of artichoke extract (AE) for 1 h and then stimulated with 1 μg/mL of lipopolysaccharides (LPS) for 3 h. Merged fluorescent images of green (CellROX dye) and DAPI (4′,6-diamidino-2-phenylindole) staining of DNA (blue) are shown. (B) Quantitative analysis of (A) shown as mean pixel intensity normalized to number of cells (#). Data bars are means ± SEMs of two independent experiments. ** p < 0.005, **** p < 0.0001.

Figure 4

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) growth in lipopolysaccharide (LPS)-stimulated THP-1 macrophages untreated (−) or treated for 24 h with 1 and 2 mg/mL of artichoke extract (AE). Cell viability is expressed as % of control (−). The value represents the means ± SEMs of three different experiments, each performed with triplicate samples.

Figure 5

Anti-inflammatory effects of artichoke extract in M1 macrophages. (A) Real Time RT-PCR assay for interleukin-6 (IL-6), Monocyte Chemoattractant Protein (MCP)-1/C-C motif chemokine ligand 2 (CCL-2) mRNA expression in human THP-1 derived macrophages untreated (−) or treated for 1 h with artichoke extract (AE) at concentrations of 1 and 2 mg/mL and then stimulated with 10 ng/mL of lipopolysaccharides (LPS) for 24 h. (B) Enzyme-linked immunosorbent assay (ELISA) for IL-6 and CCL-2 in human THP-1 derived macrophages untreated (−) or treated as in (A). The values represent the means ± SEMs of three different experiments, performed in duplicate. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001.