Walnut (Juglans regia L.) Septum: Assessment of Bioactive Molecules and In Vitro Biological Effects

Abstract

Walnut (Juglans regia L.) septum represents an interesting bioactive compound source by-product. In our study, a rich phenolic walnut septum extract, previously selected, was further examined. The tocopherol content determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed higher amounts of α-tocopherol compared to γ- and δ-tocopherols. Moreover, several biological activities were investigated. The in vitro inhibiting assessment against acetylcholinesterase, α-glucosidase, or lipase attested a real management potential in diabetes or obesity. The extract demonstrated very strong antimicrobial potential against Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella enteritidis. It also revealed moderate (36.08%) and strong (43.27%) antimutagenic inhibitory effects against TA 98 and TA 100 strains. The cytotoxicity of the extract was assessed on cancerous (A549, T47D-KBluc, MCF-7) and normal (human gingival fibroblasts (HGF)) cell lines. Flow cytometry measurements confirmed the cytotoxicity of the extract in the cancerous cell lines. Additionally, the extract demonstrated antioxidant activity on all four cell types, as well as anti-inflammatory activity by lowering the inflammatory cytokines (interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 β (IL-1β)) evaluated in HGF cells. To the best of our knowledge, most of the cellular model analyses were performed for the first time in this matrix. The results prove that walnut septum may be a potential phytochemical source for pharmaceutical and food industry.

Keywords: LC-MS/MS; anti-inflammatory activity; antimicrobial; antimutagenic; antioxidant; by-product; enzyme inhibitor; tocopherols; walnut septum.

Figure 1

The chromatographic separation of tocopherols from walnut septum extract: (1) delta-tocopherol; (2) gamma-tocopherol; (3) alpha-tocopherol.

Figure 2

Cytotoxic effect of the septum extract observed using Alamar Blue assay on A549 (A), T47D-KBluc (B), MCF-7(C) and human gingival fibroblasts (HGF) (D). The results are expressed as relative means ± standard deviations (six technical replicates for each of the three biological replicates) where the negative control (DMSO 0.2%) is 100%. Asterisks (*) indicate significant differences (p < 0.05) compared to the negative control. RFU— relative fluorescence units.

Figure 3

Antioxidant effect of the WSE evaluated using DCFH-DA (2ʹ,7ʹ-Dichlorofluorescin Diacetate) assay on A549 (A), T47D-KBluc (B), MCF-7(C) and HGF (D). The cellular model was pre-exposed to the extract (25, 40, and 50 µg/mL) or N-acetylcysteine (NAC) (20 mM) for 24 h, and further incubated with 50 µM DCFH-DA. The antioxidant effect of the WSE was evaluated after 2 h in stimulated (250 µM H₂O₂) and un-stimulated conditions. The results are expressed as relative means ± standard deviations (six technical replicates for each of the three biological replicates) where the negative control (DMSO 0.2%) is 100%. The asterisks (*) indicate significant differences compared to the positive control (250 µM H₂O₂) in stimulated conditions, while (#) indicate significant differences compared to the negative control in non-stimulated conditions (ANOVA + Tukey; p < 0.05).

Figure 4

The extracellular release of pro-inflammatory cytokines interleukin-6 (IL-6) (A), interleukin-8 (IL-8) (B) and interleukin-1 β (IL-1β) (C) was analyzed in cell-free supernatants by ELISA at 24 h post-exposure to three concentrations of the walnut septum extract (WSE) in combination with 100 ng/mL LPS (lipopolysaccharides). The values are expressed as mean ± standard deviation (SD) of four biological replicates. The asterisk (*) indicates significant differences compared to the positive control (100 ng/mL LPS), while (#) indicates a significant difference of the positive control compared to the negative control (ANOVA + Tukey; p < 0.05).