JPP 2007, 59: 1721–1728 © 2007 The Authors Received March 29, 2007 Accepted July 17, 2007 DOI 10.1211/jpp.59.12.0015 ISSN 0022-3573 1721 Antioxidant activity of 4-methylcoumarins Jens Z. Pedersen, Cristina Oliveira, Sandra Incerpi, Vineet Kumar, Anna Maria Fiore, Paolo De Vito, Ashok K. Prasad, Sanjay V. Malhotra, Virinder S. Parmar and Luciano Saso Abstract Polyphenolic coumarins are known to act as antioxidants in biological systems, but it is difficult to distinguish their antioxidant activity from the many other effects they produce in cells. We have determined the radical scavenging capacity of 22 structurally related natural and synthetic 4-meth- ylcoumarins, by measuring their reaction with radicals, galvinoxyl and 2,2-diphenyl-1-picrylhydrazyl, using electron paramagnetic resonance spectroscopy. Efficient antioxidant activity of 4-methylcou- marins in cells was verified using the DCF fluorescent probe assay for determination of intracellular reactive oxygen species levels. As expected, the o-dihydroxysubstituted coumarins were found to be excellent radical scavengers and better than the m-dihydroxysubstituted or monohydroxysubsti- tuted analogues, but surprisingly the corresponding o-diacetoxy derivatives also turned out to be good scavengers, even in the absence of an esterase. Another unexpected result was that the anti- oxidant efficiency of 4-methylcoumarins could be modulated by introducing an ethoxycarbonyl- ethyl substituent at the C-3 position; this effect cannot be explained by simple electron donating/ withdrawing properties. Coumarin concentrations of 10 mM or less were used in all experiments, cor- responding to the levels relevant for therapeutic purposes. Considering that 4-methylcoumarins, in contrast to many other coumarins, are not metabolized to toxic epoxide intermediates, these results indicate promising new strategies for the design of non-toxic antioxidant coumarin-based drugs. Coumarins are a group of compounds that show a surprising variety of biological effects. They occur naturally in many plants, fungi and bacteria, and have found applications for centuries as spices and in traditional medicine. Several natural polyphenolic coumarins show anti-inflammatory, antimicrobial, antiviral, anti-carcinogenic, anticoagulant and anti- oxidant activity; but generally the reason for these effects and the precise nature of their actions are not known (Borges et al 2005). Many different types of antioxidant activity of coumarins have been reported for a variety of biological systems, but it has not yet been possible to correlate the effects observed to the chemical structures of the coumarins studied (Fylaktakidou et al 2004; Borges et al 2005; Kostova 2006). Hydroxycoumarins are believed to behave like classic phenol- or quinol-based antioxidants, in which a hydroxy group on an aromatic ring structure can carry out the single-electron reduction of a free rad- ical. The resulting phenoxyl radical or semiquinone can either be stabilized through the presence of bulky or electron-withdrawing groups on the ring system or be oxidized further through the consecutive single-electron reduction by a second hydroxy group to produce a quinone-type end product. This is the mechanism behind both natural and synthetic antioxi- dants, like vitamin E and butylated hydroxytoluene; however, sometimes it turns out that coumarins do not work in this way (Fylaktakidou et al 2004; Kostova 2006). One problem with coumarin compounds has been the tendency to form 3,4-coumarin epoxides during metabolic degradation; these intermediates are believed to be mutagenic and probably also have other toxic effects. To prevent this problem a series of 4-methylcou- marins have been synthesized; in these compounds 3,4-coumarin epoxide formation is no longer possible as 4-methylcoumarins are not substrates for the liver P-450 monoxygenases that epoxidize coumarins lacking the C-4 methyl group. Several 4-methylcoumarins have shown promising antioxidant effects, such as inhibition of lipid peroxidation and scavenging Introduction Department of Biology, University of Rome ‘Tor Vergata’, Italy Jens Z. Pedersen, Paolo De Vito Department of Biology, University of Rome ‘Roma Tre’, Italy Sandra Incerpi, Anna Maria Fiore Department of Chemistry, University of Delhi, India Vineet Kumar, Ashok K. Prasad, Virinder S. Parmar Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, USA Vineet Kumar, Sanjay V. Malhotra Department of Human Physiology and Pharmacology, Sapienza University of Rome, Italy Luciano Saso, Cristina Oliveira Correspondence: J. Z. Pedersen, Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, 00133 Roma, Italy. E-mail: jzp@ofir.dk Acknowledgement: The financial support from the Italian Ministry for University and Research, General Management for Strategies and Development of Internationalization of Scientific and Technological Research is gratefully acknowledged. Downloaded from https://academic.oup.com/jpp/article/59/12/1721/6141796 by guest on 27 July 2022