104 B. FERRARI ET AL. Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 16: 104–107 (2005) PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 16, 104–107 (2005) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.825 Copyright © 2005 John Wiley & Sons, Ltd. Received 15 December 2003 Revised 28 April 2004 Accepted 7 May 2004 Direct Identification and Quantitative Determination of Costunolide and Dehydrocostuslactone in the Fixed Oil of Laurus novocanariensis by 13 C-NMR Spectroscopy Bernard Ferrari, 1 Paula Castilho, 2 Félix Tomi, 1 * Ana Isabel Rodrigues, 3 Maria do Ceu Costa 3 and Joseph Casanova 1 1 Université de Corse, Equipe Chimie et Biomasse, UMR CNRS 6134, Route des Sanguinaires, 20000 Ajaccio, France 2 Universidade de Madeira, Centro de Química da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal 3 INETI-DTIQ, Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada do Paço do Lumiar, P-1649-038 Lisboa, Portugal The fixed oil of Laurus novocanariensis (previously L. azorica) contains mostly glycerides together with minor non-saponifiable compounds. The direct identification and quantitative determination of costunolide and dehydrocostuslactone, two sesquiterpene lactones components of the oil that exhibit biological activities, is described. The analysis was carried out using 13 C-NMR spectroscopy (signal acquisition with inverse gated decoupling of protons; diglyme as internal standard) without separation, derivatisation or any sample prepara- tion. Copyright © 2005 John Wiley & Sons, Ltd. Keywords: 13 C-NMR; quantification; costunolide; dehydrocostuslactone; Laurus novocanariensis, L. azorica. * Correspondence to: F. Tomi, Université de Corse, Equipe Chimie et Biomasse, UMR CNRS 6134, Route des Sanguinaires, 20000 Ajaccio, France. Email: felix.tomi@univ-corse.fr Contract/grant sponsor: French CPU. Contract/grant sponsor: Collectivité Territoriale de Corse. Contract/grant sponsor: CRUP. INTRODUCTION The leaves of Laurus azorica, harvested from laurels en- demic to the macaronesian archipelagos of Madeira, Azores and Canaries, are much used in traditional medi- cine owing to attributed anti-ulcer and blood depurative properties. Recent publications have shown that these laurels exhibit anti-thrombin activity (Medeiros et al., 2000). The hexane extract of the leaves contains two lactones, costunolide (1) and dehydrocostuslactone (2) (Tinoco, 2000) that are known to possess plant growth regulatory and cytotoxic properties (Sun et al., 2003). These sesquiterpene lactones, which are mainly found in the Asteraceae but also occur infrequently in other fami- lies of higher and lower plants, have recently received renewed attention since both 1 and 2, isolated from bay leaves (L. nobilis), were found to inhibit inducible nitric oxide synthase (iNOS; Matsuda et al., 2000) and, further- more, 1 was shown to have inhibitory effects on blood- ethanol elevation (Matsuda et al., 2002), to inhibit the RAS-farnesyl-protein-transferase (Park et al., 2001), to induce differentiation in human leukaemia HL-60 cells (Choi et al., 2002a), and to trigger apoptosis in human leukaemia U937 cells (Choi et al., 2002b). Until 2002, it was considered that the laurels endemic to the archipelagos of Madeira, Azores and Canaries constituted a single species referred to as Laurus azorica (Seub.) Franco. Recently it was determined that there were sufficient distinctions to consider the laurels from Madeira and Canaries as a separated taxon, now class- ified as Laurus novocanariensis Rivas Mart., Lousã, Fern. Prieto, E. Días, J. C. Costa and C. Aguiar (Rivas- Martínez et al., 2002). On the island of Madeira, the fixed oil, traditionally obtained by boiling and crushing the fruits (drupes) of L. novocanariensis, is used in external application for its cicatrising and anti-rheumatic properties and it is taken internally for the treatment of apoplexy (Rivera and Obón, 1995). The main fatty acid chains present in the oil have been analysed by GC after trans-esterification, and were found to be oleic, palmitic, linoleic and lauric in descending order of abundance (P. Castilho, unpub- lished results). The oil also contains the sesquiterpene lactones costunolide (1) and dehydrocostuslactone (2)