Protective Effect of Cyclically Pressurized Solid⁻Liquid Extraction Polyphenols from Cagnulari Grape Pomace on Oxidative Endothelial Cell Death

Abstract

The aim of this work is the evaluation of a green extraction technology to exploit winery waste byproducts. Specifically, a solid⁻liquid extraction technology (Naviglio Extractor^®) was used to obtain polyphenolic antioxidants from the Cagnulari grape marc. The extract was then chemically characterized by spectrophotometric analysis, high-performance liquid chromatography, and mass spectrometry, revealing a total polyphenol content of 4.00 g/L ± 0.05, and the presence of anthocyanins, one of the most representative groups among the total polyphenols in grapes. To investigate potential biological activities of the extract, its ability to counteract hydrogen peroxide-induced oxidative stress and cell death was assessed in primary human endothelial cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, used to assess potential extract cytotoxicity, failed to show any deleterious effect on cultured cells. Fluorescence measurements, attained with the intracellular reactive oxygen species (ROS) probe 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA), revealed a strong antioxidant potential of the marc extract on the used cells, as indicated by the inhibition of the hydrogen peroxide-induced ROS generation and the counteraction of the oxidative-induced cell death. Our results indicate the Naviglio extraction, as a green technology process, can be used to exploit wine waste to obtain antioxidants which can be used to produce enriched foods and nutraceuticals high in antioxidants.

Keywords: Cagnulari marc; Naviglio Extractor®; endothelial cell; green extraction; oxidative stress; polyphenols.

Figure 1

HPLC analysis. Overlapping of HPLC chromatograms obtained by anthocyanin standards (red curve) and pomace extract (blue curve), wavelength = 520 nm. #1 and #2 indicate the fractions collected and further analyzed by MS.

Figure 2

MALDI-TOF MS analysis. (A) Raw extract spectrum, confirming the presence of several anthocyanins, including malvidin, malvidin-3-O-glucoside, peodinin-3-O-glucoside, and malvidin-3-(6-acetyl)-glucoside. (B) Fraction 1 and (C) Fraction 2 spectra, confirming the presence of peodinin-3-O-glucoside and malvidin-3-O-glucoside, respectively.

Figure 3

Effect of grape marc extracts on human umbilical vein endothelial cell (HUVEC) viability. (A) HUVECs were incubated for 20 h with the indicated concentrations of the extract, then cell viability was determined using the MTT assay, as reported in the materials and methods. Grape mark extract inhibits hydrogen peroxide (H₂O₂)-induced ROS generation in HUVECs. (B) HUVECs were exposed for 3 h to the indicated concentrations of grape marc extracts, and then incubated in the absence (CTRL) or presence of 75 µM H₂O₂. Fluorescence was measured, as reported, in the Materials and Methods. #, p = 0.0428; ##, p = 0.0021; ###, p = 0.001; ####, p = 0.0001. Grape mark extract inhibits hydrogen peroxide (H₂O₂)-induced oxidative cell death of HUVECs. (C) HUVECs were exposed for 3 h to the indicated concentrations of grape marc extracts and then incubate for 20 h in the absence (CTRL) or presence of 75 µM H₂O₂. Cell viability was assessed as reported in the materials and methods. Data are expressed as percentage of the control. CTRL, untreated cells; H₂O₂, hydrogen peroxide; GME, Grape marc extract. ##, p = 0.0017; ###, p = 0.0006.