Black mulberry (Morus nigra L .) prevents deleterious effects of excess glucose in obese C. elegans decreasing lipofuscin accumulation and ROS production

Abstract

Black mulberries have been traditionally used as antidiabetic agents and are a source of nutrients and phenolic compounds, particularly anthocyanins. The objective of this work is to determine if Morus nigra berries could prevent metabolic and obesity-related disorders using in vitro systems and in vivo alternative models such as C. elegans. An aqueous solvent-free extract from Morus nigra fruits rich in phenolic compounds like chlorogenic acid, hyperoside, rutin and cyanidin 3-glucoside was evaluated in the C. elegans obese model subjected to high glucose concentrations evaluating different parameters such as lipid droplets, lipofuscin accumulation and ROS production. The capacity of the extract to inhibit advance glycation end products and free radicals as well as pancreatic lipase and α-amylase was also evaluated in vitro. The black mulberry extract showed a significant capacity to inhibit the accumulation of lipid droplets, reducing by 50.40 % the fat deposits. The extract was able to reverse the deleterious effects of excess glucose in C. elegans enhancing stress resistance, preventing the accumulation of lipofuscin, and decreasing the ROS production. The anti-glycation and antioxidant effects in vitro were higher than the reference substances aminoguanidine and quercetin respectively. Morus nigra was also able to inhibit the pancreatic enzymes α-amylase and lipase and could be considered an interesting traditional food ingredient in the prevention of certain metabolic diseases.

Keywords: Anthocyanins; Diabetes; Flavonoids; Functional foods; Morus nigra; Obesity; Traditional foods.

Graphical abstract

Fig. 1

Fluorescence images, lipid droplet profile and total lipid droplet quantification of C. elegans N2 strain. A) Fluorescence images of C. elegans taken after exposing the worms to the different conditions and after staining with Nile Red and exposure to ultraviolet light; on the right, lipid droplets are highlighted. For the extract condition, only images at concentration 500 μg/mL are shown. Scale bar = 150 μm. B) Histogram showing the relative values of lipids in C. elegans obese model after being exposed to the different conditions and concentrations of M. nigra fruit extract (n = 75–100 worms). Results are expressed as mean ± SEM. ∗∗∗∗p < 0.0001 vs Glucose, ^$$p < 0.005. C) Lipid droplet average size per worm area ratio of C. elegans obese model after being exposed to the different conditions and concentrations of M. nigra fruit extract (n = 75–100 worms). Results are expressed as mean ± SEM. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs Glucose; ^$$$p < 0.0001 vs Control.

Fig. 2

C. elegans N2 strain average length in μm after exposure to different conditions for 48 h. Results are presented as mean ± SEM (n = 75–100 worms). ∗∗∗∗p < 0.0001 vs Control, ^###p < 0.001 vs Glucose.

Fig. 3

Accumulation of age pigment lipofuscin in C. elegans SS104 strain. A) Images of C. elegans autofluorescence age pigment lipofuscin taken at day 7 and day 10 of the worms being exposed to different conditions. B) Histogram with relative values of the fluorescence age pigment lipofuscin accumulation at days 7 and 10 of C. elegans being exposed to different conditions. Results are represented as mean ± SEM. ∗p < 0.05, ^#p < 0.05 vs control day 7, ^$p < 0.05 vs control day 10.

Fig. 4

Production of ROS in C. elegans SS104 strain after exposure to the different conditions for 7 and 10 days, and exposition to thermal stress (2h at 35 °C). Data are expressed as % of fluorescence in relation to control untreated worms (NGM). Results are presented as mean values ± SEM (n = 3). ∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05 Glucose vs NGM; ^##p < 0.01, ^#p < 0.05 Glucose vs Morus 500 μg/mL. Statistical significance was calculated using two-way analysis of variance ANOVA.

Fig. 5

A) Inhibition of AGEs by the M. nigra extract, aminoguanidine (AMG) used as control. B) Inhibition of free radicals by the M. nigra extract and quercetin used as control. Data are presented as mean ± SEM. (n = 3).

Fig. 6

A) Inhibition of pancreatic lipase by Morus nigra fruits, orlistat used as control. B) Inhibition of amylase by Morus nigra fruits, gallic acid used as control. Data are presented as mean ± SEM. (n = 3).