Phenolic Characterization and Neuroprotective Properties of Grape Pomace Extracts

Abstract

Vitis vinifera (grape) contains various compounds with acknowledged phytochemical and pharmacological properties. Among the different parts of the plant, pomace is of particular interest as a winemaking industry by-product. A characterization of the water extract from grape pomace from Montepulciano d'Abruzzo variety (Villamagna doc) was conducted, and the bioactive phenolic compounds were quantified through HPLC-DAD-MS analysis. HypoE22, a hypothalamic cell line, was challenged with an oxidative stimulus and exposed to different concentrations (1 µg/mL^-1 mg/mL) of the pomace extract for 24, 48, and 72 h. In the same conditions, cells were exposed to the sole catechin, in a concentration range (5-500 ng/mL) consistent with the catechin level in the extract. Cell proliferation was investigated by MTT assay, dopamine release through HPLC-EC method, PGE₂ amount by an ELISA kit, and expressions of neurotrophin brain-derived neurotrophic factor (BDNF) and of cyclooxygenase-2 (COX-2) by RT-PCR. The extract reverted the cytotoxicity exerted by the oxidative stimulus at all the experimental times in a dose-dependent manner, whereas the catechin was able to revert the oxidative stress-induced depletion of dopamine 48 h and 72 h after the stimulus. The extract and the catechin were also effective in preventing the downregulation of BDNF and the concomitant upregulation of COX-2 gene expression. In accordance, PGE₂ release was augmented by the oxidative stress conditions and reverted by the administration of the water extract from grace pomace and catechin, which were equally effective. These results suggest that the neuroprotection induced by the extract could be ascribed, albeit partially, to its catechin content.

Keywords: BDNF; COX-2; PGE2; Vitis vinifera; catechin(s); grape pomace; hypothalamus; neuroprotection; oxidative stress; water extract.

Figure 1

Chromatographic analysis of grape pomace phenolic compounds. The chromatographic analysis confirmed the presence of different phytochemicals: gallic acid (peak #1); caftaric acid (peak #2); catechin (peak #3); chlorogenic acid (peak #4); epicatechin (peak #5); caffeic acid (peak #6); syringic acid (peak #7); coumaric acid (peak #8); ferulic acid (peak # 9).

Figure 2

Effects induced by grape pomace water extract (10 mg/mL) on hearth rate in the Daphnia magna toxicity model. ANOVA, p < 0.0001; *** p < 0.001 vs. EtOH 30%, this last representing the positive control toxicity group.

Figure 3

MTT assay of HypoE22 hypothalamic cell line exposed to different concentrations (1–1000 µg/mL) of water pomace extract for 24, 48, and 72 h. The data graph bars are the mean ± SD (n = 3). • p < 0.05 vs. 48 h C; * p < 0.05 vs. 24 h H₂O₂; § p < 0.05 vs. 48 h H₂O₂; + p < 0.05 vs. 72 h H₂O_2.

Figure 4

Inhibitory effects exerted by grape pomace water extract (1–1000 µg/mL) and catechin (5–500 ng/mL) on hydrogen peroxide (H₂O₂)-induced reduction of dopamine (DA) extracellular levels (ng/mL) in HypoE22 cells. The efficacy of the extract was evaluated at different time points: 24 h (left); 48 h (middle); 72 h (right). ANOVA, p < 0.0001; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. respective hydrogen peroxide (H₂O₂) group.

Figure 5

Inhibitory effects exerted by grape pomace water extract (1–1000 µg/mL) and catechin (5–500 ng/mL) on hydrogen peroxide (H₂O₂)-induced increase in cyclooxygenase-2 (COX-2) gene expression in HypoE22 cells. The efficacy of the extract was evaluated at different time points: 48 h (A,B); 72 h (C,D). ANOVA, p < 0.0001; * p < 0.05, *** p < 0.001 vs. respective hydrogen peroxide group (H₂O₂).

Figure 6

PGE₂ production in HypoE22 hypothalamic cell line exposed to different concentrations (1–1000 µg/mL) of water pomace extract. The data graph bars are the mean ± SD (n = 3). * p < 0.05 vs. C.

Figure 7

Inhibitory effects exerted by grape pomace water extract (1–1000 µg/mL) and catechin (5–500 ng/mL) on hydrogen peroxide (H₂O₂)-induced decrease in brain-derived neurotrophic factor (BDNF) gene expression in HypoE22 cells. The efficacy of the extract was evaluated at different time points: 48 h (A,B); 72 h (C,D). ANOVA, p < 0.0001; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. respective hydrogen peroxide group (H₂O₂).