Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell lines Severina Pacifico a,1,⇑ , Marialuisa Gallicchio a,1 , Peter Lorenz b , Nicoletta Potenza a , Silvia Galasso a , Sabina Marciano a , Antonio Fiorentino a , Florian C. Stintzing b , Pietro Monaco a a Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, I-81100 Caserta, Italy b WALA Heilmittel GmbH, BadBoll/Eckwälden, Germany article info Article history: Received 18 July 2013 Accepted 24 September 2013 Available online 3 October 2013 Keywords: Laurus nobilis L. Neuroprotection Assay guided fractionation Sesquiterpenes Apoptosis Metabolic profiling abstract In the course of a bioactivity screening of Mediterranean plants, the assessment of neuroprotective prop- erties of Laurus nobilis L. was of interest. Dried leaves were extracted by sonication using CHCl 3 as solvent. The CHCl 3 parental extract (CHCl 3 -pe) was fractionated to yield CHCl 3 (LnC-1), EtOAc (LnC-2), MeOH (LnC-3) fractions. Each fraction underwent an extensive screening towards human neuroblastoma (SK-N-BE(2)-C, and SH-SY5Y) and rat glioma (C6) cell lines. MTT and SRB cytotoxicity tests were per- formed. The effect on the plasma membrane integrity was evaluated by assessment of LDH release. The caspase-3 activation enzyme and DNA fragmentation were also evaluated. The oxidant/antioxidant ability of all the extracts were evaluated using different methods. Furthermore, a metabolite profiling of the investigated extracts was carried out by GC–EI-MS. CHCl 3 -pe contained terpenes, allylphenols, and a-tocopherol. Dehydrocostus lactone was the main constituent. As result of the fractionation tech- nique, the LnC-1 extract was mainly composed of a-tocopherol, whereas the LnC-2 fraction was enriched in guaiane and eudesmane terpenes. The most cytotoxic LnC-2 fraction induced apoptosis; it was ineffec- tive in preventing in vitro free radicals production. Overall, the experimental results support a possible role of LnC-2 preparation as a chemopreventive agent for neuronal cells or other cells of the CNS. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The medicinal properties of plants are widely known to man, who, since ancient times, has learned about their beneficial and curative effects. The cumulative action exerted by the different natural constituents of a medicinal plant, or its part (plant drug), is the basis of unique pharmacological properties (beneficial and/ or adverse). Plant complex (phytocomplex) is the biochemical en- tity in which the bioactivity of the medicinal plant is expressed in its fullness. In fact, in it synergistic and additive effects, an im- proved bioavailability, or even a reduced toxicity are combined with an enhanced pharmacological efficacy, often not attributable to a single active substance (Bagetta et al., 2012). Although there are various molecular targets for which natural products have pro- ven their efficiency, biomedical research is now particularly inter- ested in defining their role in the treatment of those disorders and diseases that affect global human health, such as neurodegenera- tive diseases and brain cancer (Iriti et al., 2010; Kannappan et al., 2011). For example, malignant brain tumors are particularly aggressive in both children and adults (Li et al., 2013). Despite re- cent improvements in diagnostic techniques, therapeutic ap- proaches remain disappointing and unsuccessful. Consequently, there is an urgent need for promising anticancer agents or lead structures for the development of new drugs to raise the overall survival of patients with brain cancer. Several in vitro and in vivo studies showed the anti-brain tumor properties of some plant extracts, both as therapeutics and as com- ponents of the diet for chemoprevention (Pathak et al., 2003; Huang et al., 2007; Lantto et al., 2009). The cytotoxic effects were related to their secondary metabolites content. It was reported, for instance, that a chloroform extract from Angelica sinensis, con- taining large amount of low-molecular weight phenolics as ferulic acid, and butylidenephthalide, had the ability to inhibit growth and induce apoptosis of glioblastoma multiforme (GBM) tumor through p53-dependent and p53-independent pathways (Tsai et al., 2005; Lin et al., 2012). Grape seed extract (GSE) treatment in- duced accumulation of intracellular reactive oxygen species (ROS) in head and neck squamous cell carcinoma (HNSCC) cells leading to DNA damage causing a cell cycle arrest in G2/M phase together with cell growth inhibition and apoptotic cell death (Shrotriya et al., 2012). Moreover, chokeberry extract (Aronia melanocarpa), which is rich in anthocyanins, and curcumin from Curcuma longa were reported to possess anticancer properties in an established brain cancer cell line (Abdullah Thani et al., 2012). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.09.029 ⇑ Corresponding author. Tel.: +39 0823 274572; fax: +39 0823 274571. E-mail address: severina.pacifico@unina2.it (S. Pacifico). 1 These authors contributed equally to this research. Food and Chemical Toxicology 62 (2013) 628–637 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox