red wine extract-induced activation of endothelial nitric oxide synthase is mediated by a great variety of polyphenolic compounds

• Published in: Molecular nutrition & food research Vol. 54; no. S2; pp. S171 - S183
• Main Authors: Auger, Cyril, Chaabi, Mehdi, Anselm, Eric, Lobstein, Annelise, Schini-Kerth, Valérie B
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 01.07.2010; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Animals; Anthocyanins - chemistry; Anthocyanins - isolation & purification; Anthocyanins - pharmacology; Biochemistry, Molecular Biology; Cells, Cultured; Cellular Biology; Chemical Fractionation; Chromatography, High Pressure Liquid; Coronary Vessels - cytology; Coronary Vessels - drug effects; Coronary Vessels - metabolism; Electron Spin Resonance Spectroscopy; Endothelial function; Endothelial nitric oxide synthase; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Enzyme Activation - drug effects; Flavonoids - chemistry; Flavonoids - isolation & purification; Flavonoids - pharmacology; Fractionation; Glucosides - chemistry; Glucosides - isolation & purification; Glucosides - pharmacology; Life Sciences; Molecular Structure; Nitric Oxide - analysis; Nitric Oxide Synthase Type III - metabolism; Phenols - chemistry; Phenols - isolation & purification; Phenols - pharmacology; Phosphorylation - drug effects; Polyphenols; Proanthocyanidins - chemistry; Proanthocyanidins - isolation & purification; Proanthocyanidins - pharmacology; Proto-Oncogene Proteins c-akt - metabolism; Red Wine; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sus scrofa; Wine - analysis
• ISSN: 1613-4125; 1613-4133
• DOI: 10.1002/mnfr.200900602

Phenolic extracts from red wine (RWPs) have been shown to induce nitric oxide (NO)-mediated vasoprotective effects, mainly by causing the PI3-kinase/Akt-dependent activation of endothelial NO synthase (eNOS). RWPs contain several hundreds of phenolic compounds. The aim of the present study was to identify red wine phenolic compounds capable of activating eNOS in endothelial cells using multi-step fractionation. The red wine phenolic extract was fractionated using Sephadex LH-20 and preparative RP-HPLC approaches. The ability of a fraction to activate eNOS was assessed by determining the phosphorylation level of Akt and eNOS by Western blot analysis, and NO formation by electron spin resonance spectroscopy. Tentative identification of phenolic compounds in fractions was performed by MALDI-TOF and HPLC-MS techniques. Separation of RWPs by Sephadex LH-20 generated nine fractions (fractions A to I), of which fractions F, G, H and I caused significant eNOS activation. Fraction F was then subjected to semi-preparative RP-HPLC to generate ten subfractions (subfraction SF1 to SF10), all of which caused eNOS activation. The active fractions and subfractions contained mainly procyanidins and anthocyanins. Isolation of phenolic compounds from SF9 by semi-preparative RP-HLPC lead to the identification of petunidin-O-coumaroyl-glucoside as a potent activator of eNOS.