Black Mulberries (Morus nigra L.) Modulate Oxidative Stress and Beta-Amyloid-Induced Toxicity, Becoming a Potential Neuroprotective Functional Food

Abstract

Black mulberry (Morus nigra L.) is a common edible fruit from the Moraceae family with a wide variety of nutritional and medicinal applications, mainly due to its antioxidant and anti-inflammatory properties. The purpose of this work was to investigate the cytoprotective and neuroprotective capacity of a hydrophilic black mulberry solvent-free extract rich in polyphenols, including the antioxidant, antiradical, and enzymatic mechanisms that would explain these effects. Its neuroprotective potential was evaluated in vitro using the Neuro-2a cell line and in vivo through the Caenorhabditis elegans organism model. Neuro-2a cells were treated at different concentrations of the extract (25-500 µg/mL) and hydrogen peroxide (300 µM) as an oxidant agent, simultaneously. From these treatments, redox status (intracellular ROS production) and cellular activity (MTT) were also quantified in Neuro-2a. Regarding the C. elegans assay, the protection of the extract against β-amyloid toxicity was measured against the CL4176 strain, which is a model of Alzheimer disease. As a complementary neuroprotective assay, its potential to inhibit the monoamine oxidase A (MAO-A) enzyme was measured. In addition, an Artemia salina bioassay was performed for preliminary toxicity screening. And its antioxidant properties were evaluated by means of the FRAP assay. The results confirm its neuroprotective potential and its ability to scavenge free radicals and decrease ROS production, also acting as a moderate MAO-A inhibitor. Moreover, the polyphenolic extract alleviates the toxicity induced by β-amyloid accumulation in C. elegans. Concluding, Morus nigra can be considered a functional food with bioactive compounds that may prevent the onset of neurodegenerative diseases.

Keywords: ROS; antioxidant activity; black mulberry; functional foods; neuroprotection; phenolic compounds.

Figure 1

Most common Morus species. (A) Morus rubra. (B) Morus nigra. (C) Morus alba.

Figure 2

Mitochondrial activity in Neuro-2a cells (MTT assay). (A) Cytotoxicity in Neuro-2a cells after exposure to different concentrations of black mulberry extract (Morus nigra). Note: **** p < 0.0001 and *** p < 0.001 compared to control. Differences calculated using one-way ANOVA. (B) Cytoprotective effect of black mulberry extract on Neuro-2a cells against hydrogen peroxide. Note: **** p < 0.0001, *** p < 0.001 and ** p < 0.01 compared to hydrogen peroxide. ### p < 0.001 compared to control. Differences calculated using one-way ANOVA. (C) Microscope images of different concentrations compared to the control. Differences in toxicity of the extract (300, 400, 500 µg/mL Neuro-2a apoptosis).

Figure 3

ROS production in Neuro-2a cells subjected to oxidative stress by hydrogen peroxide (300 μM) and treatment with blackberry extract (25, 50, 100 and 200 μg/mL). The data are expressed as a percentage of the control cells. Note: **** p < 0.0001, ** p < 0.01 and * p < 0.05 versus hydrogen peroxide. #### p < 0.0001 versus control; ns: not significant. Two-way ANOVA was used as statistical analysis.

Figure 4

Effect of M. nigra extract on paralysis curves in C. elegans CL4176. At least 150 worms in three replicates were studied per condition. PT50 was 28 h for the control group and 32 h for worms treated with the extracts. The results of the paralysis assay were analyzed using the Kaplan–Meier survival model and for statistical significance by using a log-rank pairwise comparison test.

Figure 5

Non-toxic effect of black mulberry on Artemia salina. Significant differences were observed between control and 500 µg/mL treatment in Metanauplius stage (* p < 0.05). Significant differences were calculated through ANOVA and Dunnett’s Multiple Comparison Test.

Figure 6

MAO−A inhibition by Morus nigra and Clorgyline. IC₅₀ = 0.023 µg/mL and 49.64 µg/mL for Clorgyline and black mulberry extract, respectively.