Model Study of the Enzymatic Modification of Natural Extracts: Peroxidase-Based Removal of Eugenol from Rose Essential Oil Charfeddine Bouhlel, Gwenn’Ann Dolhem, Xavier Fernandez, and Sylvain Antoniotti* LCMBA UMR 6001 CNRS - Universite ́ de Nice-Sophia Antipolis, Institut de Chimie de Nice, Parc Valrose, 06108 Nice cedex 2, France * S Supporting Information ABSTRACT: A protocol based on the use of horseradish peroxidase (HRP) is proposed for the removal of allergenic eugenol from rose essential oil without loss of the organoleptic quality and with a good conservation of the chemical composition. For the first time, an enzyme-based strategy is proposed for essential oils treatment and opens new opportunities in the detoxification of natural extracts used in perfumery and cosmetics. Our results on eugenol in rose essential oil constitute a first step toward the development of efficient and mild processes for the removal of more toxic compounds of natural extracts. KEYWORDS: HRP, enzymatic remediation, natural extracts, fragrances, allergens ■ INTRODUCTION Natural extracts were the first type of material used in perfumery with incenses of ancient oriental civilizations and are still of paramount importance in modern fragrances formulas. Precious materials such as essential oils from rose, sandalwood, patchouli, and vetiver among others are used in almost every fine fragrance. 1,2 These extracts are typically composed of several dozens of individual compounds with a great qualitative and quantitative variability, most of them being of terpenoid origin. 3 In spite of an ancestral use for many of these, the need of consumers and authorities of Western countries for absolutely benign products on the market could in a near future be a threat on a fair number of natural extracts. To date, the presence of allergens belonging to the list of 26 has to be specified on the final manufactured products when they exceed minimum levels. A series of putative carcinogens such as methyleugenol, 4,5 safrole, 6,7 skin sensitizers such as atranol derivatives (contained by tree moss extracts which are listed among the 26), 8,9 or hepatotoxic suspected agents such as estragole are also under close surveillance. 10 To anticipate the plausible ban of certain natural materials containing these compounds, research efforts are currently done in many laboratories, both in academia and industry, to find efficient processes allowing for the selective removal of these suspected toxic compounds. A critical issue for such processes is to avoid the alteration of the organoleptic quality of the material treated. The main type of process for this purpose is distillation, which suffers from weak selectivity and high energy demand. Others solutions have been evaluated such as laser photolysis, 11 or trapping on specific solid supports such as molecular imprinted polymers, 12 but have not yet been applied to such processes so far. In such context, biocatalysis could offer both the specificity, the most famous asset of enzyme activity, and the mildness required to handle sensitive materials such as essential oils. In addition to these practical advantages, biocatalysis could also provide more sustainable chemical processes, compared to the conventional time- and energy-consuming distillation strategies, operating with selectivity issues. In this paper is presented a model study of the enzymatic removal of eugenol from rose essential oil, one of the most precious materials of fine perfumery. This study is the first example of enzyme-assisted modification of essential oils aimed at removing a single compound and constitutes a proof-of-concept series of experiments demonstrating that such strategy could be viable in the future for more challenging toxic compounds. ■ MATERIALS AND METHODS Chemicals. Solvents (Et 2 O, CH 2 Cl 2 ), salts (NaH 2 PO 4 , Na 2 HPO 4 ), and eugenol were purchased from Aldrich and used as received. Horseradish peroxidase (HRP) was purchased from Sigma, and catalase was from Fluka, stored at -18 °C, and let warm to room temperature prior to use. A rose essential oil communelle (blending of oils of various origins) was used in this study. Samples of dieugenol 13 and diacetyldieugenol 14 could be prepared following procedures from the literature. Apparatus. 1 H NMR and 13 C NMR spectra were recorded on a Bruker AC 200. 1 H NMR spectra are described as follows: chemical shift (δ) in ppm relative to TMS at 0 ppm, multiplicity (s = singlet, d = doublet, m = multiplet), coupling constants (Hz), and integration. 13 C NMR spectra chemical shifts are reported in ppm (δ) relative to CDCl 3 at 77.16 ppm. Fast-GC/MS analyses were performed for routine analyses with a Shimadzu QP2010S-MS chromatograph (EI, 70 eV) equipped with an SLB-5 ms capillary column (thickness, 0.10 μm; length, 15 m; inside diameter, 0.10 mm). Temperature program: 80 °C then 18°/min to 200 °C and maintained at this temperature for 30 min. Analytical thin-layer chromatography (TLC) was performed on 0.2 mm precoated plate silica gel 60 F254 (Merck). Quantitative Gas Chromatography Analysis. GC analysis was carried out using an Agilent 6890N gas chromatograph, under the following operation conditions: vector gas, helium; injector and detector temperatures, 250 °C; injected volume, 1 μL; split ration 1/ Received: August 10, 2011 Revised: December 27, 2011 Accepted: December 30, 2011 Published: December 30, 2011 Article pubs.acs.org/JAFC © 2011 American Chemical Society 1052 dx.doi.org/10.1021/jf205194v | J. Agric.Food Chem. 2012, 60, 1052-1058