Research paper 4-Methylcoumarins as antioxidants: Scavenging of peroxyl radicals and inhibition of human low-density lipoprotein oxidation Fausta Natella a, * , Bénédicte Lorrain a , Ashok K. Prasad b , Virinder S. Parmar b , Luciano Saso c , Cristina Scaccini a a National Research Institute on Food and Nutrition, INRAN, Via Ardeatina 546, 00178 Rome, Italy b Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India c Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, Italy article info Article history: Received 12 January 2010 Accepted 22 April 2010 Available online 29 April 2010 Keywords: 4-methylcoumarins Antioxidant activity Low density lipoprotein Structureeactivity relationship abstract The antioxidant activity of eight synthetic 4-methylcoumarins was systematically studied. The antioxi- dant capacity was measured using: (i) a competition kinetic test, to measure the relative capacity to quench peroxyl radical; (ii) the in vitro oxidative modification of human low-density lipoprotein, initiated by AAPH or catalyzed by copper. In both models, the ortho-OH substitutes were found to be better antioxidant than the meta one. The most efficient antioxidant was the 7,8-dihydroxy-4-methylcoumarin and the corresponding diacetoxy-substituted was unexpectedly a good antioxidant. Finally, the presence of an ethoxycarbonylethyl substituent at the C-3 position increased the antioxidant capacity of both 7,8-dihydroxy-4-methylcoumarin and 7,8-diacetoxy-4-methylcoumarin. Ó 2010 Elsevier Masson SAS. All rights reserved. 1. Introduction A huge number of studies accumulated in the past decade support the oxidative hypothesis of atherosclerosis, in which the oxidation of LDL represents the key event of atherosclerosis development [1e5]. Ox-LDL has indeed a wide range of atherogenic properties, from the formation of early lesions to plaque rupture [6]. For this reason, the inhibition of LDL oxidation by naturally or synthetic antioxidants has been considered as a possible pharmacological strategy to prevent atherosclerosis and cardiovascular disease. Coumarins comprise a large class of compounds found throughout the plant kingdom and displaying multiple biological properties that could promote human health. Chemically, couma- rins consist of fused benzene and a-pyrone rings; because of substitutions, which can occur at any of the six available sites, more than 1300 coumarins have been identified from natural sources [7]. Coumarins have been largely studied in vivo and in vitro for their biological activities and some natural or synthetic derivatives have found pharmaceutical applications. Several coumarins have shown anti-inflammatoy, anticarcinogenic, antiviral, antithrombotic, anti- allergic, hypolipidaemic and antioxidant activities [7]. However, the biochemical mechanisms at the basis of these effects are not clearly known. Some of the pharmacological activities of coumarins could be ascribed to their capacity to inhibit lipoxygenase and cyclo- xygenase [8,9] and to scavenge reactive oxygen species [10,11]. The antioxidant activities of coumarins and their metabolites and/or derivatives have been reported in many biological systems. As for the biological activity, the antioxidant activity of these compounds is strongly influenced by their chemical structure. Unsubstituted coumarins can form 3,4-coumarin epoxides during their metabolism and these intermediates could be toxic [12]. Differently, synthetic 4-methylcoumarins do not induce the formation of the epoxide, as they are not substrates for liver P450 monoxygenase [12]. For this reason, they could be better candidates for pharmacological use. As coumarins, 4-methylcoumarins are known to have some pharmacological effects, such as anti- inflammatory activity [8]. 4-methylcoumarins have shown to possess antioxidant activity, and several studies have been reported on structureeantioxidant activity relationship of these compounds [13,14]. However, the great part of these studies analyze the anti- oxidant activity of 4-methylcoumarins toward ‘not physiological’ radicals (such as DPPH) [15,16] or in ‘not physiological’ system (such as linoleic acid lipid peroxidation) [17]. Only one study reports on structure/antioxidant activity relation of three 4- methylcoumarins in LDL oxidation model (oxidation was initiated Abbreviation: AAPH, 2,2 0 -Azobis(2-amidinopropane) dihydrochloride; DAMC, Diacetoxy-4-methylcoumarins; DHMC, Dihydroxy-4-methylcoumarin; DPPH, 1,1- Diphenyl-2-picrylhydrazyl radical; LDL, Low density lipoprotein; MC, Methylcoumarin. * Corresponding author. Tel.: þ39 6 51494447; fax: þ39 6 51494550. E-mail address: natella@inran.it (F. Natella). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.04.019 Biochimie 92 (2010) 1147e1152