J. of Supercritical Fluids 55 (2011) 1007–1013 Contents lists available at ScienceDirect The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu Processing cherries (Prunus avium) using supercritical fluid technology. Part 2. Evaluation of SCF extracts as promising natural chemotherapeutical agents Ana Teresa Serra a , Ana A. Matias a,b,c , Ana P.C. Almeida b , M.R. Bronze a,b,c , Paula M. Alves a,b , Hermínio C. de Sousa d , Catarina M.M. Duarte a,b,∗ a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Estac ¸ ão Agronómica Nacional, 2780-157 Oeiras, Portugal b Instituto de Biologia Experimental e Tecnológica, Avenida da República, Quinta-do-Marquês, Estac ¸ ão Agronómica Nacional, Apartado 12, 2781-901 Oeiras, Portugal c IMED, Faculdade de Farmácia da Universidade de Lisboa, Av. das Forc ¸ as Armadas, 1649-019, Lisboa, Portugal d CIEPQPF, Chemical Engineering Department, University of Coimbra, Pólo II, Rua Sílvio Lima, 3030-790 Coimbra, Portugal article info Article history: Received 31 December 2009 Received in revised form 6 June 2010 Accepted 16 June 2010 Keywords: Antiproliferative Cell cycle arrest Cherry Supercritical fluid extraction Perillyl alcohol abstract In the last years, there has been a growing interest in the recovery of bioactive compounds from natural sources for the development of novel functional foods. In this study, natural extracts, previously obtained by fractioned high pressure extraction from “Saco” cherry culls, were characterized in terms of anticancer activity. The product derived from CO 2 :EtOH (90:10, v/v) extraction after a pre-treatment of raw material with supercritical CO 2 during 1 h, exhibited the highest antiproliferative activity in human HT29 colon cancer cells. In addition, when compared with doxorubicin, cherry extracts induced cell cycle arrest in a different cell cycle checkpoint. In order to obtain extracts with enhanced antiproliferative activity, the extraction process was further explored. Using highly pure CO 2 and EtOH 96% the inhibition of cancer cell growth was significantly enhanced by 16-fold. In addition, the incorporation of a conventional extraction step with MetOH or EtOH:H 2 O (50:50, v/v) prior to fractioned process allowed to obtain cherry extracts more concentrated in antiproliferative compounds. Perillyl alcohol present in cherry extracts was pointed to be one of the major responsible for anticancer properties of cherry extracts. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Cancer is one of the most leading causes of death worldwide. According to a recent report by the World Health Organization (WHO), the disease accounted for 7.9 million deaths (or around 13% of all deaths worldwide) in 2007 and the projection is that this number will increase up to 18 million in 2020 [1]. In particular, colorectal cancer is the third most common form of cancer in men (after prostate and lung) and the second in women (after breast). Diet and lifestyle are pointed to be major risk factors for developing this type of cancer [2]. Epidemiological data suggest that ingestion of bioactive com- pounds from fruits and vegetables, such as polyphenols and terpenes, may contribute to reduce the incidence of cancer in humans [3]. The mechanisms by which these compounds inhibit tumourgenesis include inhibition of tumour cell mediated protease ∗ Corresponding author at: IBET – Instituto de Biologia Experimental e Tecnológ- ica, Apartado12, 2781-901 Oeiras, Portugal. Tel.: +351 21 446 97 27; fax: +351 214421161. E-mail address: cduarte@itqb.unl.pt (C.M.M. Duarte). activity [4], attenuation of tumour angiogenesis [5], induction of cell cycle arrest [6–8] and promotion of apoptosis [9,10]. In addi- tion, it has been reported that combining natural compounds with chemotherapeutic agents is a promising strategy to enhance the inhibition of tumour survival [11,12]. Sweet cherry (Prunus avium) is a much appreciated fruit by con- sumers due to its taste, colour and sweetness. Moreover, cherries constitute a rich source of bioactive compounds, namely flavonoids (anthocyanins, flavan-3-ols and flavonols) [13,14], that are already reported to exhibit anticancer properties [15–17]. In particular, cherry contains perillyl alcohol [3], a hydroxylated monocyclic monoterpene, which has been shown to have a potential use as an anticancer drug by stimulating different types of tumours to apop- tosis [18,19], inhibiting their proliferation [20–22] or overcoming their resistance to chemo-/radiotherapy [23,24]. Therefore, peril- lyl alcohol has been investigated by the National Cancer Institute (NCI) in NCI-sponsored phase I, II or III chemoprevention trials for prostate, breast and colon cancer [25]. “Saco” cherry is an old traditional Portuguese variety that has protected geographical indication (PGI) registration according to the EU regulations (“Cova da Beira” cherry). In spite of the pow- erful antioxidant and antiproliferative effect demonstrated for this 0896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2010.06.006