J. of Supercritical Fluids 47 (2008) 37–48 Contents lists available at ScienceDirect The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu Fractioned SFE of antioxidants from maritime pine bark Mara E.M. Braga a,∗ , Rosa M.S. Santos b , Inês J. Seabra a,b , Roselaine Facanali c , Márcia O.M. Marques c , Hermínio C. de Sousa a a CIEPQPF, Chemical Engineering Department, University of Coimbra, 3030-790 Coimbra, Portugal b ESAC, Politechnical Institute of Coimbra, Bencanta, 3040-316 Coimbra, Portugal c Laboratório de Produtos Naturais, Centro de P&D Recursos Genéticos Vegetais, Instituto Agronômico de Campinas, Campinas, Brazil article info Article history: Received 30 October 2007 Received in revised form 22 May 2008 Accepted 29 May 2008 Keywords: Maritime pine bark Antioxidants Fractioned supercritical fluid extraction CO2 Ethanol abstract Maritime pine (Pinus pinaster) is an important portuguese forest species and its bark is an abundant furniture industry residue. In this work, fractioned supercritical fluid extraction (FSFE) experiments were carried out using CO 2 and CO 2 +EtOH (10%) mixtures in two consecutive steps. Different pres- sures (from 10 up to 30MPa) and temperatures (30, 40 and 50 ◦ C) were assayed. FSFE extracts were compared with hydrodistillation (HD) and Soxhlet (SoE) extracts. Gas chromatography (GC) was used to characterize the obtained volatile oils while catechin and epicatechin were quantified in ethanolic extracts by HPLC. Antioxidant activities were determined spectrophotometrically. Around 84% of the total extract was obtained in the 1st CO 2 extraction step and this volatile oil rich extract presented lower oxidation inhibitions (∼29–62%) than those obtained at the 2nd CO 2 + EtOH extraction step (60–84%). The catechin + epicatechin yields of these 2nd step ethanolic extracts were higher (0.051–0.346 g/mg d.b.) than the ones obtained for SoE (0.039 g/mg d.b.). The increment in catechin + epicatechin extract content influenced the corresponding extract antioxidant activity, although not directly. Pressure and temperature affected the extraction kinetic parameters for both extraction steps and the obtained cate- chin + epicatechin contents for the 2nd CO 2 + EtOH extraction step. The employed high pressure fractioned extraction methodology showed that it was possible to obtain different extract fractions having diverse antioxidant capacities from Pinus pinaster bark. Furthermore, this process can be further optimized in order to improve extraction yields, selectivities and antioxidant activ- ities, by the use of different solvent mixtures compositions and of other different operational conditions. Therefore, the reuse of this abundant portuguese agroindustrial residue may originate several possible high-value extracts for different applications like, for example, the use the volatile oil fractions in the aroma/flavor industries, and/or the use of phenolic fractions in the food and pharmaceutical industries. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Maritime pine (Pinus pinaster) is one of the most important portuguese native forest species which comprises approximately 23% of all the portuguese forest area and achieved an annual pro- duction of 671,000 tonnes during the year 2006 [1]. Considering that, for southern pines of pulpwood size, bark volumes are usually between 12% and 24% of total produced pine volume (Koch, 1972, as cited by Gao et al. [2]), there are huge amounts of pine bark residues which are readily available at low prices. Therefore, these residues can be used as a raw material following the worldwide tendency of recovering, recycling and upgrading wastes. When compared to other common residues generated by the portuguese ∗ Corresponding author. Tel.: +351 239 798 758; fax: +351 239 708 703. E-mail address: marabraga@eq.uc.pt (M.E.M. Braga). agricultural activities, like grape pomace, pine bark presents some important advantages such as its all year availability, its low price, its long-term stability and its easy way to handle and process [3]. In addition, and due to its high polyphenolic content, P. pinaster bark (which is mostly used as firewood for energy production) has also a strong potential to be used as a raw material for the produc- tion of high-value bioactive substances. The most common phenolic compounds present in maritime pine bark are (+)-catechin, (-)-epicatechin, dihydroquercetin and numerous phenolic acids, most of them being procyanidin dimers, trimers, oligomers and polymers [4], i.e., proanthocyanidins (or commonly known as condensed tannins). Like other pine barks, P. pinaster bark also contains fatty acids, aliphatic acids, resin acids, sterols, as well as other lipophilic compounds [5] which have been reported to present antioxidant activity and also to cause synergis- tic effects with other antioxidants [6]. 0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2008.05.005