Antioxidant and Antisenescence Effects of Bergamot Juice

Abstract

Aging is one of the main risk factor for the onset of cardiovascular diseases; one of the possible explanations could be linked to the age-associated overproduction of free radicals. This increase of oxidative stress can be overcome with a high intake of food antioxidants. In this context, a number of studies have been addressed to assess the antiaging potential of natural antioxidant compounds. Recently, it has been shown that the juice of bergamot (Citrus bergamia Risso et Poiteau), a fruit mostly produced in the Ionian coastal areas of Southern Italy (Calabria), is a valuable source of health-promoting constituents with, among other, antioxidant properties. In order to investigate the potential antiaging effects of this Mediterranean natural antioxidant source, bergamot juices of three different cultivars ("fantastico," "femminello," and "castagnaro") were herein characterized by the mean of high-performance liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry. Then, juices were investigated for the evaluation of total polyphenolic and flavonoid contents, cell-free model antioxidant activities, and in vitro antiaging properties on two different cellular models of induced myocardial senescence. The best performing juice was also assessed in vivo. The phytochemical profiles confirmed that juices were rich in flavonoids, both flavone and flavanone glycosides. In addition, two limonoid glycosides were also identified in all cultivars. Each cultivar showed different phenolic and flavonoid contents. In tube results showed the juice robust antioxidant activities that correlate with their phenolic and flavonoid contents. Moreover, for the first time, the ability of juice to counteract the chemical-induced senescence was here demonstrated in both cellular models. Lastly, the in vivo data obtained from mouse hearts evidenced an increase in transcription of genes involved in antiaging and antioxidant responses. The overall results suggest that bergamot juice exerts antioxidant and antisenescence effects, making it useful for nutraceutical purposes.

Figure 1

HPLC-PDA profiles of C. bergamia juices from “fantastico” (a), “femminello” (b), and “castagnaro” (c) cultivars. Peaks were monitored at 325 nm. For the peak data, see Table 2.

Figure 2

ESI-MS/MS spectra of compounds 6 and 7. ESI-MS/MS spectra of compound 6 at m/z 901 (a) and compound 7 at m/z 885 (b), performed in the negative ion mode. For the peak data, see Table 1.

Figure 3

RACI values obtained for the C. bergamia cultivars. RACI values were obtained comparing the total phenolic content, the superoxide radical scavenging activity, the ferric reducing antioxidant power, and the lipid peroxidation inhibition results of investigated cultivars.

Figure 4

Effects of C. bergamia “fantastico” cultivar juice on the mitochondrial oxidative metabolism activity. H9c2 cells were treated with three different concentrations of lyophilized juice, dissolved in DMSO, and the mitochondrial oxidative metabolism activity was examined.

Figure 5

Effects of C. bergamia “fantastico” juice on H9c2 senescence-associated β-galactosidase staining. (a) Representative phase contrast photomicrographs of control cells, DOX-injured cells, and DOX-injured cells treated with FBJ, 0.1 mg/mL. The arrows indicate the blue-stained cells. (b) Percentage of cellular senescence in not-injured cells treated with different concentrations of FBJ. Data are shown as the percentages of β-galactosidase-positive cells with respect to the total cell number of the sample. Each bar represents the mean ± SEM of three replicates from three independent experiments. (c) Percentage of cellular senescence in H₂O₂- or DOX-injured cells treated with FBJ at different concentrations. Data are shown as the percentages of β-galactosidase-positive cells with respect to the total cell number of the sample. Each bar represents the mean ± SEM of three replicates from three independent experiments. The light gray bars represent the data obtained from the H₂O₂-induced senescence model; the dark gray bars represent the data obtained from the DOX-induced senescence model. ^∗∗∗p < 0.001 versus the control (cells not injured); °°°p < 0.001 versus the H₂O₂- or DOX-challenged cells with DMSO; °°p < 0.01 versus the H₂O₂- or DOX-challenged cells with DMSO; °p < 0.05 versus the H₂O₂- or DOX-challenged cells with DMSO.

Figure 6

FBJ induces gene expression. Real-time PCR analyses showed a statistically significant increase in SIRT1, NRF2, and FOXO3 mRNA levels in hearts of old mice fed with FBJ for three months. ^∗p < 0.05; ^∗∗∗p < 0.005.

Figure 7

FBJ induces NQO1 and HO-1 gene expression. Real-time PCR analyses showed a statistically significant increase in NQO1 and HO-1 mRNA levels in hearts of old mice fed with FBJ for three months. ^∗∗p < 0.01; ^∗∗∗p < 0.005.