Mehdi Chaabi

The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on hydroxyl moieties.

Several rich sources of polyphenols stimulate the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase (eNOS). The present study examined the molecular mechanism underlying the stimulatory effect of epicatechins on eNOS. NO-mediated relaxation was assessed using porcine coronary artery rings in the presence of indomethacin, and charybdotoxin plus apamin, inhibitors of cyclooxygenases and EDHF-mediated responses, respectively. The phosphorylation level of Akt and eNOS was assessed in cultured coronary artery endothelial cells by Western blot, and ROS formation using dihydroethidine. (-)-Epigallocatechin-3-O-gallate (EGCg) caused endothelium-dependent relaxations in coronary artery rings and the phosphorylation of Akt and eNOS in endothelial cells. These responses were inhibited by membrane-permeant analogues of superoxide dismutase and catalase, whereas native superoxide dismutase, catalase and inhibitors of major enzymatic sources of reactive oxygen species including NADPH oxidase, xanthine oxidase, cytochrome P450 and the mitochondrial respiration chain were without effect. The EGCg derivative with all hydroxyl functions methylated induced neither relaxations nor the intracellular formation of ROS, whereas both responses were observed when the hydroxyl functions on the gallate moiety were present. In conclusion, EGCg causes endothelium-dependent NO-mediated relaxations of coronary artery rings through the Akt-dependent activation of eNOS in endothelial cells. This response is initiated by the intracellular formation of superoxide anions and hydrogen peroxide, and is critically dependent on the gallate moiety and on the presence of hydroxyl functions possibly through intracellular auto-oxidation.

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

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.

Activity-Guided Isolation of Antioxidant Principles from Limoniastrum feei (Girard) Batt.

Abstract Bioguided fractionation of a leaves extract from Limoniastrum feei (Girard) Batt. (Plumbaginaceae) led to the isolation of seven polyphenolic constituents: gallic acid (1), myrciaphenone A (2), myricetin-3-O-β-galactopyranoside (3-1), epigallocatechin gallate (3-2), myricetin 3-O-α-rhamnopyranoside (4), quercetin (5) and myricetin (6). Gallic acid was the most antioxidant compound in DPPH [(0.94 d 0.68) μg/mL] and FRAP [(0.83 d 0.15) μm Fe2+/mL] tests, whereas myricetin was a more specific superoxide radical scavenger since it was the most active product in the superoxide nitroblue tetrazolium hypoxanthine/xanthine oxidase test [(1.86 d 0.12) μg/mL].

Antimalarial compounds from the stem bark of Vismia laurentii.

A phytochemical study of the stem bark of Vismia laurentii resulted in the isolation of a tetracyclic triterpene, tirucalla-7,24-dien-3-one (1), and seven other known compounds: 3-geranyloxyemodin (2), vismiaquinone A (3), vismiaquinone B (4), bivismiaquinone (5), epifriedelinol (6), betulinic acid (7) and stigmasta-7,22-dien-3-ol (8). The structure of all these compounds was elucidated by spectroscopic means. The stem bark extract and compounds 1 and 3 showed good antimalarial activity against the W2 strain of Plasmodium falciparum.

A new flavonoid and other constituents from Centaurea nicaeensis All. var. walliana M.

A new flavone glucoside, apigenin 4′-(6″-methylglucuronide) (1), together with six known compounds, cirsilineol, jaceosidin, melitensin, apigenin, apigenin 7-(6″-methylglucuronide) and prunasin, were isolated from the ethanolic extract of the aerial parts of Centaurea nicaeensis All. var. walliana M. (Asteraceae) collected from Souk-Ahras, eastern Algeria. The structures were established by spectral analysis, mainly HRESI-MS, UV and 2D-NMR experiments (COSY, HSQC and HMBC).

C007 Redox-sensitive activation of endothelial nitric oxide synthase by catechins: role of hydroxyl moieties

Objectives Several rich sources of polyphenols have been shown to strongly increase the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase. The purpose of the present study was to investigate the molecular mechanisms underlying the stimulatory effect of catechins on the endothelial formation of NO using different catechins (flavan-3-ols). Methods Vascular reactivity studies were performed using porcine coronary artery rings, which were suspended in organ chambers for the measurement of changes in isometric tension. All experiments were performed in the presence of indomethacin (an inhibitor of cyclooxygenases), and the combination of apamin and charybdotoxin (two inhibitors of endothelium-derived hyperpolarizing factor-mediated effects) to assess only the NO component of the relaxation. Cultures of porcine coronary artery endothelial cells (P1) were used to determine the phosphorylation level of Akt and endothelial NO synthase by Western blot analysis. Both natural and synthetic catechins were evaluated. Results (-)-Epigallocatechin-3-O-gallate (EGCg) induced potent endothelium-dependent relaxations in porcine coronary artery rings. The EGCg-induced relaxation was inhibited by MnTMPyP (a membrane permeant analogue of superoxide dismutase, SOD) whereas extracellular SOD had no effect, indicating a major role of the intracellular formation of ROS. Relaxations to EGCg were minimally affected by rotenone (an inhibitory of the mitochondrial respiratory chain), sulphenazol (an inhibitor of cytochrome P450), apocynin (an inhibitor of NADPH oxidase) or allopurinol (an inhibitor of xanthine oxidase). The replacement of all hydroxyl groups of EGCg by O-methyl groups resulted in the total loss of the relaxing activity whereas partial replacement decreased the relaxing activity. Conclusions EGCg caused endothelium-dependent relaxations of coronary arteries via the redox-sensitive formation of NO in endothelial cells. The stimulatory effect does not involve major intracellular sources of ROS including the mitochondrial respiratory chain, xanthine oxidase, NADPH oxidase and cytochrome P450 but is critically dependent on the presence of hydroxyl groups possibly leading to auto-oxidation of the polyphenol.

C008 Endothelial NO synthase activation by a red Wine Phenolic extract: isolation of active components by bioguided fractionation

Objectives Previous studies have shown that Red Wine Phenolic extracts induce nitric oxide (NO)-mediated vasoprotective effects, mainly by causing a redox-sensitive activation of endothelial NO synthase (eNOS). However, Red Wine Phenolic extracts are complex mixtures of a several hundreds of phenolic compounds. Therefore, the aim of the present study was to isolate active phenolic compounds using multi-step bioguided fractionation of the red wine extract. Methods Red Wine Phenolic Extract was submitted to a bioguided fractionation using chromatographic methods. The fractions obtained at each step were tested for their potential to induce the activation of eNOS in endothelial cells. Cultures of porcine coronary artery endothelial cells (P1) were used to determine the phosphorylation level of Akt and endothelial NO synthase by Western blot analysis. Identification of phenolic compounds in each active fraction was performed by MALDI-TOF and HPLC-MS techniques. Results The first step of fractionation on lipophilic Sephadex® yielded 9 fractions of which 4 of them significantly increased the phosphorylation level of Akt and eNOS in endothelial cells. The active fractions contained mainly procyanidins and some anthocyanins compounds. The fractionation of one of the active fractions by preparative reverse-phase HPLC yielded 11 subfractions ; all of these sub-fractions significantly increased the phosphorylation level of Akt and eNOS. The analysis of the phenolic compounds indicated that these sub-fractions contained mixtures of procyanidin dimers or conjugated anthocyanins. Conclusions The red wine extract contains several types of phenolic compounds, which are able to enhance the activity of NO synthase in endothelial cells including procyanidins dimers and oligomers as well as several conjugated anthocyanins.