Polyphenol-rich strawberry extract protects human dermal fibroblasts against hydrogen peroxide oxidative damage and improves mitochondrial functionality

Abstract

Strawberry bioactive compounds are widely known to be powerful antioxidants. In this study, the antioxidant and anti-aging activities of a polyphenol-rich strawberry extract were evaluated using human dermal fibroblasts exposed to H2O2. Firstly, the phenol and flavonoid contents of strawberry extract were studied, as well as the antioxidant capacity. HPLC-DAD analysis was performed to determine the vitamin C and β-carotene concentration, while HPLC-DAD/ESI-MS analysis was used for anthocyanin identification. Strawberry extract presented a high antioxidant capacity, and a relevant concentration of vitamins and phenolics. Pelargonidin- and cyanidin-glycosides were the most representative anthocyanin components of the fruits. Fibroblasts incubated with strawberry extract and stressed with H2O2 showed an increase in cell viability, a smaller intracellular amount of ROS, and a reduction of membrane lipid peroxidation and DNA damage. Strawberry extract was also able to improve mitochondrial functionality, increasing the basal respiration of mitochondria and to promote a regenerative capacity of cells after exposure to pro-oxidant stimuli. These findings confirm that strawberries possess antioxidant properties and provide new insights into the beneficial role of strawberry bioactive compounds on protecting skin from oxidative stress and aging.

Figure 1

Viability of Human Dermal fibroblast (HuDe) determined by MTT assay after incubation with different concentrations of strawberry extract and at different times.Data are expressed as mean ± SEM for eight replicas (n = 8) of three independent experiments.

Figure 2

Viability of Human Dermal fibroblast (HuDe) determined by MTT assay after incubation with H₂O₂. Control and strawberry pre-treated cells (0.5 mg/mL) were stressed with various concentrations of H₂O₂ (0–50 mM). Cells with extract differ significantly from controls for concentrations of H₂O₂ between 0.5 and 5 mM. Data are expressed as mean ± SEM for eight replicas (n = 8) of three independent experiments. * p < 0.05 significantly different from control.

Figure 3

Levels of ROS following H₂O₂-induced stress. Control and strawberry extract pre-treated cells were incubated with H₂DCFDA and then stressed with different concentrations of H₂O₂ (0–10 mM). The kinetics of fluorescence was studied for two hours. Variation of fluorescent signal is significant already after 15 min of stress for H₂O₂ ≥ 5 mM and after 30 min for concentrations of 2.5 mM. Results are reported as mean DCF fluorescence activity (arbitrary units) obtained from at least three separate experiments (error bars represent SEM). * p < 0.05 significantly different from control, ** p < 0.01 highly significant compared to control.

Figure 4

Evaluation of lipid peroxidation after H₂O₂ stress. Control and pre-treated cells with extract were incubated with BODIPY for 30 min and stressed with different concentrations of H₂O₂ (0–500 μM). The relationship between red and green fluorescence over time decreases significantly for concentrations of 5 μM hydrogen peroxide and highly significantly for concentrations ≥ 50 μM compared to controls. Data are expressed as mean ± SEM for eight replicas (n = 8) of three independent experiments. * p < 0.05 significantly different from control, ** p < 0.01 highly significant.

Figure 5

Evaluation of oxygen consumption rate (OCR) in control and in strawberry pre-treated fibroblasts, stressed with H₂O₂. OCR was monitored through Seahorse XF-24 Extracellular Flux Analyzer with the sequential injection of Oligomycin (1 µg/mL), 2,4-DNP (100 µM), Rotenone (1 µM) at the indicated time point into each well, after baseline rate measurement (A). Basal OCR levels in control, strawberry pre-treated fibroblasts and in cells stressed with H₂O₂ with and without pretreatment with the strawberry extract (B).

Figure 6

Comet assay after H₂O₂ stress. Cells were stressed with H₂O₂ for 15 min, processed and analysed for DNA damage. The data are expressed as the percentage of intensity fluorescence in the comet tail (percentage tail intensity) in control and pre-treated cells and relate to three independent experiments performed in triplicate. Statistical analysis was performed with T-test. * p < 0.05 significantly different from control.

Figure 7

MTT and recovery after stress by H₂O₂. Control and pre-incubated cells were stressed with different concentrations of H₂O₂ (0.1, 0.5 and 1 mM) and analysed in different times to assess the percentage of vitality. At 0 and 24 h after stress, the difference from controls was significant for concentrations of hydrogen peroxide ≥ 500 mM. After 72 h the two curves differed for all the experimental points. Data are expressed as mean ± SEM for eight replicas (n = 8) of three independent experiments. Statistical analysis was performed with the T-test. * p < 0.05 significantly different from control, ** p < 0.01 highly significantly.