To evaluate the antioxidant activity of different phenolic acids and their esters, three types of experiments have been used. Electron paramagnetic resonance (EPR) quantitative analysis was carried out using the acetaldehyde/xanthine oxidase system and Fenton's reaction to generate superoxide and hydroxyl radicals, respectively. In a second test, hydroperoxides generated by Cu2+-catalysed oxidation of low density lipoproteins (LDL) were quantified by a modified iodometric method. In a third assay, LDL were oxidized with Esterbauer's method and modified LDL species were quantified by HPLC. The results show that the esterified phenolic derivatives present a better antioxidant activity, on the lipoperoxidation of LDL, than the corresponding phenolic acids

Abella, Annie

Chalas, Jacqueline

Labidalle, Serge

Lindenbaum, Albert

Monje, Marie-Carmen

Nepveu, Françoise

Souchard, Jean-Pierre

Urizzi, Pascale

Vergnes, Laurent

International audienceTo evaluate the antioxidant activity of different phenolic acids and their esters, three types of experiments have been used. Electron paramagnetic resonance (EPR) quantitative analysis was carried out using the acetaldehyde/xanthine oxidase system and Fenton's reaction to generate superoxide and hydroxyl radicals, respectively. In a second test, hydroperoxides generated by Cu2+-catalysed oxidation of low density lipoproteins (LDL) were quantified by a modified iodometric method. In a third assay, LDL were oxidized with Esterbauer's method and modified LDL species were quantified by HPLC. The results show that the esterified phenolic derivatives present a better antioxidant activity, on the lipoperoxidation of LDL, than the corresponding phenolic acids

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Antioxidant activity of phenolic acids and esters present in red wine on human Low-Density Lipoproteins

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       Open Archive Toulouse Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of some Toulouse researchers and makes it freely available over the web where possible.   This is an author's version published in: https://oatao.univ-toulouse.fr/23186     Official URL : https://doi.org/10.1051/jcp:1999117   To cite this version :            Any correspondence concerning this service should be sent to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr   Urizzi, Pascale and Monje, Marie-Carmen  and Souchard, Jean-Pierre and Abella, Annie and Chalas, Jacqueline and Lindenbaum, Albert and Vergnes, Laurent and Labidalle, Serge and Nepveu, Françoise  Antioxidant activity of phenolic acids and esters present in red wine on human Low-Density Lipoproteins. (1999) Journal de Chimie Physique et de Physico-Chimie Biologique, 96 (1). 110-115. ISSN 0021-7689 OATAO Open Archive Toulouse Archive Ouverte Antioxidant activity of phenolic acids and esters present in red wine on human low-density lipoproteins P. Urizzi 1 , M.-C. Monje1 , J.-P. Souchard1 , A. Abella2, J. Chalas2, A. Lindenbaum2, L. Vergnes 1, S. Labidalle1 and F. Nepveu1: 1 Laboratoire de Synthèse, Physico-Chimie et Radiobiologie, Université Paul Sabatier,35 chemin des Maraichers, 31062 Toulouse cedex 4, France 2 Service de Biochimie, Hôpital Antoine Bée/ère, 92141 Clamart, France• Correspondence and reprints. RÉSUMÉ Trois expériences ont été menées afin d'évaluer l'activité antioxydante de différents acides et de leurs esters. Une analyse quantitative par résonance paramagnétique électronique (RPE) a été réalisée en utilisant le système acétaldéhyde/xanthine oxydase et la réaction de Fenton générant, respectivement, les radicaux superoxyde et hydroxyle. Dans un second test, les hydroperoxydes générés par une réaction d'oxydation des lipoprotéines de basse densité (LOL) catalysée par Cu2+ ont été quantifiés par une méthode iodométrique modifiée. Dans une troisième étude, les LOL ont été oxydées par la méthode d'Esterbauer et les espèces oxydées ont été quantifiées par HPLC. Les résultats montrent que les dérivés estérifiés présentent une activité antioxydante contre la lipoperoxydation des LOL bien plus importante que celle des acides phénoliques correspondants. Mots-clé : antioxydants, composés phénoliques, lipoprotéines, vin rouge. ABSTRACT To evaluate the antioxidant activity of different phenolic acids and their esters, three types of experiments have been used. Electron paramagnetic resonance (EPR) quantitative analysis was carried out using the acetaldehyde/xanthine oxidase system and Fenton's reaction to generate superoxide and hydroxyl radicals, respectively. In a second test, hydroperoxides generated by Cu2+ -catalysed oxidation of low density lipoproteins (LOL) were quantified by a modified iodometric method. In a third assay, LOL were oxidized with Esterbauer's method and modified LDL species were quantified by HPLC. The results show that the esterified phenolic derivatives present a better antioxidant activity, on the lipoperoxidation of LDL, than the corresponding phenolic acids. Key words : antioxidants, phenolic compounds, lipoproteins, red wine. INTRODUCTION Severa} lines of evidence implicate oxidatively damaged LDL as atherogenic agents. By activating inflammatory events, oxidized lipoproteins may contribute to all stages of the atherosclerotic process [1]. Because atherosclerotic vascular disease is the leading cause of death among persons with a western life-style, there has been much interest in recent years in the possible effects of antioxidants in retarding oxidative modifications of LDL. The "French Paradox", i.e. a relatively low incidence of cardiovascular events in spite of diets high in satured fat, is attributed to the regular drinking of red wine [2]. Since most of the phenolic acids contained in wine are present as ester derivatives, the aim of our work was to compare the antioxidant capacity of both chemical forms. Three tests were carried out, one using the EPR method and spin trapping technique, the second measuring hydroperoxides after the Cu2+ -catalysed oxidation of LDL and the third based on the determination of oxidized LDL by HPLC. MATERIAL AND METHODS Reagents Xanthine oxidase, 5,5-dimethyl-lpyrroline-N-oxide (DMPO) used as spin trap were obtained from Sigma Chemical Co.; gallic, caffeic, ferulic and sinapic acids were from Aldrich Chemical Co.; acetaldehyde, ethylenediaminetetraacetic acid (EDTA), H2O2 were from Prolabo. DMPO was purified and prepared as reported elsewhere [3]. Synthesis of ethanolic esters was performed as previously described [ 4]. In vitro EPR tests Hydroxyl radical scavenging tests A 1 ml solution was prepared in a glass tube by adding the reagents in the following order : DMPO (50 m.M), H2O2 (100 mM), EDTA (104 µM), phenolic compound (104M), FeSO4 (100 µM). For more details see [5].  Superoxide radical scavenging tests A 1 ml solution was prepared in a glass tube by adding the reagents in the following order : DMPO (50 m.M), xanthine oxidase (18 mU/mL), phenolic compounds (104M) and acetaldehyde (60 mM) in phosphate buffer (pH 7.4). For more details see [6]. LDL peroxidation tests Native LDL were isolated from human plasma [7). The effects of phenolic compounds on the susceptibility of LDL to oxidation were investigated by determining the hydroperoxide concentration after the Cu++-catalysed oxidation of LDL. A kinetic adaptation of El Saadani' s iodometric method was used [8]. LDL oxidation tests LDL were oxidized with Esterbauer's method [9] by exposure to 1.6 µM copper ions (Cu++) in phosphate buffer for 3-8 h at 25°C. This procedure generate three LDL species, then separated by HPLC [10). RESULTS AND DISCUSSION In order to estimate the antioxidant activity of phenolic acids and their esters, three methods were used. For the EPR studies the contrai signals were obtained from the Fenton or acetaldehyde/xanthine oxidase reaction with DMPO as spin trap. The % inhibition of the intensity of the HO- or 02- spin adduct signals were determined after adding, successively, gallic acid, caffeic acid, ferulic acid, sinapic acid, ethyl gallate, ethyl caffeate, ethyl ferulate and ethyl sinapate ( 104 M) (Fig. 1 a, 1 b ). Considering the HO· test, ethyl ferrulate presents a higher antioxidant capacity than the other compounds tested. When the 02- test was carried out at a 104 M concentration of the tested compound, caffeic acid or ethyl caffeate led to the same % inhibition of the EPR signal (100%, Fig. l b). When the concentration of these two tested compounds was decreased to 10-5 M, the % inhibition of the EPR signal was still 100% for ethyl caffeate and.. 61±5% for caffeic acid. Taking into account both tests, an eight tested compounds, except ferulic acid, presented a higher scavenging activity against 02- than against HO·. = 1� l J= ·==° 1"' D � □ � ... • • r:ri 0 1 � = -� 1� l 1 u 1 u �I -:c -= 1 .5 ':!e. • � Fl 1 2 3 4 l' 2' 3' 4' Figure 1 : Antioxydant activity of phenolic acids and esters determined by the inhibition of the signal intensity of hydro:,;yl (a) and superoxide (b) DMPO-adduct EPR signais with gal/ic acid (]), caffeic acid (2), ferulic acid (3), sinapic acid (4), ethyl gallate (l '), ethyl caffeate (2 '), ethyl -0 5 ::1. -Q,} ,:, -�0 Q,} C. 0 -0 >, ::c���-• �lL 10 ' 1 1 5 feru/ate (3 ') and ethyl sinapate (4 '). ■ l1 021 ■ l'1 02'10 4000 3000 2000 1000 0 J�jl � 1 1 1 5 @3 l'JJ4 ml4'm 1 10 Concentration (µM) Concentration (µM) Figure 2 : Concentration of hydroperoxides after LDL oxidation as a function of the phenolic compound added (gallic acid (1), caffeic acid (2), ferulic acid (3), sinapic acid (4), ethyl gallate (l '), ethyl caffeate (2 '). ethyl ferulate (3 ') and ethyl sinapate (4 ')).- -- ---- -;- - - --.- - - . . . . - . ~ '- ~ ... . -· --In a second test, the protective effect of phenolic acids and esters on the susceptibility of LDL to oxidation, was determined by measuring hydroperoxides formed by copper-mediated oxidation using the iodometric method [8). At a concentration of 10 µM, caffeic and sinapic acids, their esters and ferulic ester delayed the LDL peroxidation (Fig. 2). In a third assay, LDL were oxidized with Esterbauer's method. The Apo B oxidation increased the negative charge of the protein leading to three LDL fractions A, B, C, which can be separated by HPLC using an anion exchange column. The phenolic esters ( 10 µM) presented a better protective effect against oxidation than the corresponding acids, except ethyl gallate (Fig. 3). 120 �100 80 60 40 .2 20 0 nLDL oLDL 2 3 4 I' 2' 3' 4' Figure 3 : Pourcentage of the different LDL HP LC fractions (A : • B : &, C : cp as a fonction of the phenolic compaund added (gallic acid (1), caf eic acid (2), ferulic acid (3), sinapic acid (4), ethyl galiote (1 '), ethyl caffeate (2 '), ethyl ferulate (3 ') and ethyl sinapate (4 '), native WL (nWL), axidiud WL (oLDL)). These observations are consistent with the EPR tests (Fig. 1 ). However, in this third test, the sinapic acid prevents LOL from oxidation although no effect was observed with the EPR analysis. In conclusion, phenolic esters appeared more active than their corresponding acid forrns in our experimental conditions. Ethyl caffeate and ethyl ferulate presented the highest antioxidant activities. V, C: REFERENCES 1 Berliner J.A., Heinecke J.W. (1996) Free Radical Bio/. Med. 20, 707-727. 2 Renaud S., De Lorgeril M. (1992) Lancet 339, 1523-1526. 3 Souchard J.-P., Massol M., Nepveu F. (1996) J. Chim. Phys. 93, 214-219. 4 Gaind K.N., Juneda T.R. (1962) Jndian J. Pharm. 24, 316-318. 5 Souchard J.-P., Limasset B., Michel F., Crastes de Paulet A., Labidalle S., Nepveu F. (1995) C. R. Soc. Bio!. 189, 1171-1181.6 Arnaiz Garcia F.J., Capu! C., Castan P., Deguenon D., Derache P., Nepveu F. (1990) Metal Ions in Biology and Medecine, John Libbey Eurotext, Paris, 21 p. 7 Havel R.J., Eder H.A., Bragdon J.H. (1995) J. Clin. Jnvest. 34, 1345-1353. 8 El Saadani, Esterbauer H., El Sayed M., Gober M., Nasser A.Y., Jurgens G. (1989) J. Lipid Res. 30, 627-630.9 Esterbauer H., Gebicki J., Puhl H., Jurgens G. (1992) Free Rad. Bio/. Med. 13, 341-390.10 Vedie B., Myara l., Pech M.A., Maziere J.C., Maziere C., Caprani A., Moatti N. (1991) J. Lipid Res. 32, 1359-1369. 

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Antioxidant activity of phenolic acids and esters present in red wine on human Low-Density Lipoproteins

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